Psychotropic agents having glutamate NMDA activity

ABSTRACT

The invention provides novel compounds and pharmaceutical compositions for the treatment of psychological and/or psychiatric diseases or disorders.

This application is a national phase entry of International ApplicationNo. PCT/IL2007/001296, filed Oct. 25, 2007, which claims the benefit ofU.S. Provisional Application No. 60/854,091, filed Oct. 25, 2006.

FIELD OF THE INVENTION

The present invention is generally in the field of pharmaceuticalcompositions and methods for the treatment of psychological and/orpsychiatric diseases or disorders.

BACKGROUND OF THE INVENTION

Glutamate is the most abundant excitatory amino acids in the centralnervous system (CNS). Glutamate acts through a number of receptors thataffect both fast and slow neurotransmission. The fast ionotropicN-methyl-D-aspartate (NMDA), the amino-3-hydroxy-5-methyl-4-isoxazole(AMPA), and the slow metabotropic kainate receptor are a series ofG-protein coupled receptors. The ionotropic NMDA receptors areexcitatory which has binding sites for glutamate and glycine, andpossess an important role in memory and in mood disorders. Themetabotropic kainate glutamate receptors (mGluRs) are present at bothpresynaptic and postsynaptic sites and are considered major targets inthe area of neuropharmacology, including schizophrenia, depression,learning, memory, anxiety, seizures, addiction to drugs,neurodegeneration and developmental regulation of synaptic circuits.

The anti-psychotic drugs are widely used in the treatment of centralnervous system (CNS) psychotic diseases and disorders, such asschizophrenia. These agents block generally dopamine receptors and aredivided into typical and atypical classes; phenothiazines are, forexample, typical antipsychotics and clozapine, olanzapine andrisperidone are classified as atypical antipsychotics. It is well knownin the art that typical neuroleptic agents induce extrapyramidalsymptoms, which include rigidity, tremor, bradykinesia (slow movement),and bradyphrenia (slow thought), as well as tardive dyskinesia, acutedystonic reactions and akathasia. Atypical antipsychotics cause minimalextrapyramidal symptoms and thus rarely cause tardive dyskinesias,akathasia or acute dystonic reactions. The administration of atypicalantipsychotic agents involves other side effects such as increase ofbody weight, mood disturbances, sexual disfunction, sedation,orthostatic hypotension, hypersalivation, lowered seizure threshold and,in particular, agranulocytosis.

Schizophrenia is a chronic, debilitating disease with significantmorbidity and mortality that often requires antipsychoticpharmacotherapy for life. Current therapy consists of neuroleptics ofthe typical and the atypical type which share a common anti-dopaminergicactivity. In recent years, evidence has been accumulated suggesting thatschizophrenia and bipolar disorders are also associated with disturbancein GABA and glutamate transmission in the brain. Recent studies suggestthat schizophrenia is associated with NMDA receptor pathology. Thishypothesis is based on the experimental finding that agents that blockNMDA receptors such as phencyclidine (PCP) and MK-801 induce psychosessimilar to that associated with schizophrenia. Post-mortem data inbrains of schizophrenic patients showed also a decrease in theexpression of several glutamate receptor subtypes including NMDA, AMPAand Kainate in different brain areas. Since hypo function of the NMDAsystem is considered to have an important role in schizophrenia, andschizophreniform psychosis caused by PCP resembles schizophreniaespecially in negative symptoms and cognitive dysfunction, it wassuggested that NMDA inhibition would lead to diminished GABAergic tone,which in turn will induce disinhibition of glutamatergic AMPA receptorresulting in excitotoxic neuronal damage and psychosis.

Since direct-acting NMDA agonists, such as glutamate, might beneurotoxic, research focused on assessing the therapeutic activity ofpartial or full agonists on the glycine (GLY) site of the NMDAreceptors. Agents like D-serine and D-cycloserine (DCS) showed in someclinical studies an improvement of mainly primary negative symptoms ofschizophrenia when used as adjuvants to conventional neuroleptics suchas risperidone and olanzapine. Sarcosine is a glycine transporter 1inhibitor found efficacious in improving symptoms (negative andpositive) of patients with stable chronic schizophrenia. Thedisadvantage, however, associated in the use of these amino acids liesin the fact that they scarcely penetrate the blood brain barrier (BBB).

Glutamate receptors subtype antagonists, agonists and partial agonistsare target of intensive research. The NMDA receptor antagonist memantinewas developed for the treatment of Alzheimer's disease. The partialagonist agent D-cycloserine was found to induce some antidepressant andanxiolytic activity in animal models and to improve mood, insomnia andappetite. It is suggested that its anxiolytic effect is related toincreased learning and fear extinction. Yet, a significantantidepressant activity of D-cycloserine compared to placebo in men wasnot observed. Furthermore, agents targeting both the ionotrophic and themetabotrophic receptors of glutamate are under different stages ofdevelopment for the treatment of anxiety, depression, cognitive andmotor disorders.

SUMMARY OF THE INVENTION

The present invention discloses novel Central Nervous System (CNS)active compounds, such as psychotropic agents, having anti-dopaminergicactivity and ability to modulate glutamate N-methyl-D-aspartate (NMDA)receptor activity. Such agents are useful in the treatment ofschizophrenia and bipolar depression, and in particular have the abilityto alter the negative symptoms of schizophrenia. Such novel agents arealso useful in altering states of other mood disorders such asdepression and anxiety, cognitive deficits, movement disorders and drugaddiction.

In one aspect of the present invention, there is provided a CNS activecompound (herein a compound of the invention) conjugated to a modulatorof the glutamate NMDA receptor. The CNS compound conjugated as recitedherein, as known to a person skilled in the art, is a therapeutic agentthat acts at a site within the central nervous system, particularlywithin the brain. CNS-active compounds include CNS depressants, CNSstimulants, and drugs that selectively modify CNS function, such asanticonvulsants, anti-Parkinsonian drugs, opioid and non-opioidanalgesics, appetite suppressants, anti-emetics, analgesic-antipyretics,certain stimulants, antidepressants, antimanic agents, antipsychoticagents, sedatives and hypnotics.

Within the scope of the present invention, the CNS-active agents are notlimited to agents that act solely within the central nervous system.

In one embodiment, the compound of the invention, or a salt, a prodrug,or a stereoisomer thereof, is of the general formula L-M-V, wherein

-   -   L is a CNS active moiety;    -   M is a linker; and    -   V is a modulator of the glutamate NMDA receptor.

It should be noted that within the scope of the present invention, theCNS active agent when part of a compound of the invention is referred toas a “CNS active moiety”, signifying its conjugation to the linker, M,or the modulator of the glutamate NMDA receptor V.

The compound of the formula L-M-V may be schematically exemplified asshown:

Linker M may be conjugated to the CNS active moiety, L, via any atom ofthe CNS active moiety. The conjugation to L may be through one or moreof the native atoms of the CNS active compound, namely through one ormore atoms selected, for example, amongst C, N, S, P, and O, of the CNSactive compound (in its unconjugated form), or through a chemicallymodified part of the CNS active compound, provided that such amodification (necessary for the attachment of the linker M) does notalter or diminish the activity associated with the CNS active compound.

Linker M may be conjugated to the modulator of the glutamate NMDAreceptor, V, through any atom of the linker. As the modulator istypically an amino acid or an amino acid derivative, conjugation to theamino acid or amino acid derivative is typically through the α-carbonatom of the amino acid or amino acid derivative, as demonstrated below.

In cases where chemical modification of the CNS active compound and/orthe modulator is required to enable bonding of the linker moiety, M,thereto (to either or both L and V), the modification may be any suchmodification known to a person skilled in the art. Based on thedescription provided herein, the artisan would know how to chemicallymodify a CNS active compound for conjugation. For general syntheticmethodologies, see for example Comprehensive Organic Transformations: AGuide to Functional Group Preparations, Richard C. Larock, 2^(nd) Ed.,John Wiley & Sons, Inc., 1999.

In some embodiments, the bond between M and L and/or between M and V isnon-hydrolysable, namely the linker M does not dissociate from eitherthe CNS active moiety and/or the modulator of the glutamate NMDAreceptor, V, under aqueous physiological conditions. In someembodiments, the bonds between M and L and/or M and V are covalent.

In some embodiments, only one of the bonds between M and L or M and V isa non-hydrolizable bond while the other is a hydrolizable bond.

In other embodiments, both the bonds between M and L and between M and Vare non-hydrolizable.

As stated above, the CNS active moiety may be any one moiety derivedfrom a CNS active compound selected from the classes typically referredto as CNS depressants, CNS stimulants, and drugs that selectively modifyCNS function, such as anticonvulsants, anti-Parkinsonian drugs, opioidand non-opioid analgesics, appetite suppressants, antiemetics,analgesic-antipyretics, stimulants, antidepressants, antimanic agents,antianxiety agents, antipsychotic agents, sedatives and hypnotics.

In some embodiments, the CNS active moiety is selected from ananti-depressant compound, an anti-psychotic compound, an anti-epilepticcompound, anti-anxiety and a compound for treating a movement disorder.

In one embodiment, the CNS active moiety is derived from ananti-depressant compound selected amongst anti-unipolar agents andanti-bipolar agents. Non-limiting examples of anti-unipolar agents arefluoxetine, fluvoxamine, desipramine, paroxetine and sertraline.

Non-limiting examples of anti-bipolar agents are remoxipride,alizapride, clozapine, olanzapine and quetiapine.

In another embodiment, the CNS active moiety is derived from ananti-psychotic compound selected from clozapine, olanzapine, quetiapine,loxapine, risperidone, flupenthixol, thioridazine, chlorpromazine,perphenazine, fluphenazine, zuclopenthixol, spiperone, amisulpride,sulpiride, remoxipride and alizapride.

In yet another embodiment, the CNS active moiety is derived from ananti-anxiety compound selected from fluoxetine, fluvoxamine,desipramine, paroxetine and sertraline.

In some other embodiments, the CNS active moiety is derived from a CNSactive compound selected amongst monocyclic, bicyclic and tricyclicanti-psychotic agents.

Non-limiting examples of monocyclic agents are amisulpride, sulpiride.Non-limiting examples of bicyclic agents are spiperone, remoxipride,alizapride. Non-limiting examples of tricyclic agents arechlorpromazine, perphenazine, fluphenazine, zuclopenthixol, clozapine,olanzapine, quetiapine, loxapine, flupenthixol and thioridazine.

In certain embodiments, the CNS active moiety is derived from clozapine,olanzapine, or quetiapine.

Within the scope of the present invention, the expression “CNS activemoiety derived from” signifies the conjugation of a CNS active compound,as defined and exemplified, to afford a conjugate form of the compound,namely a conjugate moiety having the linker conjugated thereto. Forexample, a CNS active moiety derived from clozapine is a compound of theinvention in which L is clozapine, M is a linker conjugated toclozapine, and V is a modulator conjugated to M.

The linker M may or may not be present.

In some embodiments, where M is absent, L is conjugated directly to V.

In other embodiments, M is present and is typically a linear groupconjugating L and Y through one or more atoms on each moiety. The lineargroup is typically a chain of between 1 and 8 atoms having at least oneatom selected from C, N, O and S.

In some embodiments, M is selected from —NH—, —NH₂ ⁺—, —O—, —S—,C₁-C₈-alkylene, C₃-C₈-cycloalkylene, —CH₂—O—CH₂, —(CH₂), —O—(CH₂)_(n)—,—(CH₂—O)—, and —(CH₂CH₂—O)_(n)—, wherein said alkylene and cycloalkylenemay optionally be substituted by one or more groups selected from C₁-C₄alkyl, C₂-C₄ alkenyl, and C₂-C₄ alkynyl, and wherein each of n,independently of each other, is an integer between zero and 3 (i.e.,being 0, or 1, or 2, or 3). The alkylene or cycloalkylene may beinterrupted by at least one heteroatom selected from N, O and S or by atleast one or more double or triple bond.

Non-limiting examples of a linker are —NH—, —O—, —S—, methylene,ethylene, propoylene, isopropylene, isobutylene, sec-butylene,tert-butylene, butylenes, pentylene, isohexylene, hexylene, heptylene,octylene, —(CH₂—CH═CH—CH₂)—, —(CH═CH₂—CH₂—CH₂)—,—(CH₂—CH═CH—CH₂—CH₂—CH₂)—, —(CH₂—C═C—CH₂)—, —(C═C—CH₂—CH₂)—,—(CH₂—NH—CH═CH—CH₂)—, —(CH₂—NH—CH₂—CH₂—CH₂)—, —(CH₂—O—CH₂—CH₂)—,(CH₂)_(n)—, —CH₂—O—CH₂, —(CH₂—O)—, —(CH₂CH₂—O)_(n)— wherein n is aninteger between 0 and 3, and substituted derivatives thereof.

The modulator of the glutamate NMDA receptor, V, is typically an aminoacid, or ester, or amide, or alkylated amine of said amino acid. In someembodiments, the amino acids are selected from glycinyl (derived fromglycine), sarcosinyl (derived from sarcosine), serinyl (derived fromserine) and cysteinyl (derived from cysteine). In other embodiments, theamino acids are esters or amides of glycinyl, sarcosinyl, serinyl andcysteinyl.

In further embodiments, the modulator of the glutamate NMDA receptor is(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740)or a derivative thereof.

Non-limiting examples of esters of the above amino acids are C₁-C₆esters such as methyl, ethyl, propyl, butyl and hexyl esters ofglycinyl, sarcosinyl, and serinyl. The amides may be of C₂-C₆ acids (ofthe general formula C₁-C₆—COOH, wherein the C₁-C₆ carbon moiety attachedto the —COOH, from which the amide is derived, is an alkyl havingbetween 1 and 6 carbon atoms).

It is to be understood that the compounds provided herein may containone or more chiral centers. Such chiral centers may be of either the (R)or (S) configuration, or may be a mixture thereof. Thus, the compoundsprovided herein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. In the case of the amino acid moietiesconstituting the functional moieties of the glutamate NMDA receptor,either the L- or D-form may be present. As used herein, the term “aminoacid” refers to α-amino acids which are racemic, or of either the D- orL-configuration.

It is also to be understood that the chiral centers of the compoundsprovided herein may undergo epimerization in vivo. As such, one ofskilled in the art will recognize that administration of a compound inits (R) form is equivalent, for compounds that undergo epimerization invivo, to administration of the compound in its (S) form.

The term “alkylene” as used herein refers to an alkyldiyl functionalgroup having two free valences carbon atoms, namely an alkyl groupsubstituted at both ends. The expression “C₁-C₈ alkylene” refers to suchan alkyl group having between 1 and 8 carbon atoms. The term“cycloalkylene” similarly refers to a cyclic alkyl being substituted atboth ends.

Additionally, as may be known to a person skilled in the art, the term“C₁-C₄ alkyl” refers to an aliphatic chain of between 1 and 4 carbonatoms, being substituted at one position only. The term “C₂-C₄ alkenyl”refers to a carbon chain having between 2 and 4 carbon atoms and atleast one C—C bond being a double bond. The term “C₂-C₁ alkynyl” refersto a carbon chain having between 2 and 4 carbon atoms and at least oneC—C triple bond.

In certain embodiments of the invention, the CNS active compound is atricyclic anti-psychotic agent of the general formula (A) and the CNSactive moiety is derived therefrom:

wherein

X is selected from —NH—, —O— and —S—;

Y is selected from —C═C—, —NH—, —O—, and —S—;

Z and Z′ are each independently selected from C₁-C₄ alkyl and halide (I,Br, Cl and F). In some cases, each of the rings substituted by Z or Z′may be substituted by one or more of Z and/or Z′.

In some embodiments, X is —NH— or S and Y is —C═C— or S. In otherembodiments, where X is —NH—, Y is —C═C— or —S—. In other embodiments,where X is S, Y is —C═C—.

In further embodiments, Z is a methyl group. In still furtherembodiments, Z′ is a halide.

In further embodiments, the tricyclic compound of formula (A) isselected from olanzapine, quetiapine, and clozapine, the structures ofwhich are shown below and the CNS active moiety is derived fromolanzapine, quetiapine, or clozapine:

These compounds may be conjugated to a linker through any one of theircarbon or heteroatoms (i.e., S, N or O). As an example, olanzapine maybe conjugated to a linker via any of the atoms shown with an arrow:

In some embodiments, where the CNS active moiety is derived fromolanzapine, the compound of the general formula L-M-V is a compound offormula (I):

wherein M and V are as defined above.

In some embodiments, the modulator of the glutamate NMDA receptor, V, isglycinyl, or an ester thereof, and the compound is of the formula (Ia):

wherein in the compound of general formula (Ia):

M is selected from null, —NH—, —O—, —S—, C₁-C₈-alkyl, C₃-C₈-cycloalkyl,—CH₂—O—CH₂—, —(CH₂—O)_(n)—, and —(CH₂CH₂—O)_(n)—,

n is an integer between 0 and 3, and

R is selected from H and a C₁-C₄ alkyl.

In some embodiments of formula (Ia), the linker is absent and exemplarycompounds of the invention are herein designated Compounds 1 andCompound 2.

In some other embodiments of formula (Ia), the linker contains at leastone heteroatom selected from N, O and S.

In some embodiments, the linker is a C₁-C₅-alkylene containing at leastone heteroatom.

In some further embodiments, the C₁-C₅-alkylene is interrupted by an Oatom and exemplary groups are —CH₂—O—CH₂, —(CH₂—O)_(n)—, and—(CH₂CH₂—O)_(n)—, wherein n is an integer between 1 and 3 (i.e., 1 or 2or 3).

Exemplary compounds of such a structure of formula (Ia) are thecompounds herein designated as Compound 3 and Compound 4.

In certain embodiments of general formula (I), the modulator of theglutamate NMDA receptor is sarcosinyl, or an ester thereof or serinyl,or an ester thereof, and the compound is of formula (Ib):

wherein in the compound of formula (Ib):

M is selected from null, —NH—, —O—, —S—, C₁-C₈-alkyl, C₃-C₈-cycloalkyl,—CH₂—O—CH₂—, —(CH₂—O)_(n)—, and —(CH₂CH₂—O)_(n)—,

n is an integer between 0 and 3,

R and R′, independently of each other are selected from H and aC₁-C₄-alkyl, and R″ is selected from H and —CH₂OH.

In some embodiments, where R′ is a C₁-C₄-alkyl, R″ is H and R isoptionally different from H.

In some embodiments, where R′ is H, R″ is —CH₂OH.

In other embodiments, in the compound of formula (Ib), the linker M isabsent, R″ is H and R′ is selected from H and C₁-C₄-alkyl. Exemplarycompounds of the invention are herein designated Compound 5 throughCompound 10.

In other embodiments of formula (Ib), the linker, M, is C₁-C₅-alkylenecontaining a heteroatom selected from N, O and S.

In some embodiments, the linker is a C₁-C₅-alkylene interrupted by atleast one O atom, such as —CH₂—O—CH₂, —(CH₂—O)_(n)—, and—(CH₂CH₂—O)_(n)—, wherein n is as defined above. Exemplary compounds areherein designated as Compound 11 through Compound 13.

Similarly to Compounds 1 through 13, which are based on olanzapine asthe CNS active moiety, compounds of the general formula L-M-V which arebased on clozapine and quetiapine have also been prepared. Table 1 listsan exemplary selection of compounds according to the invention. In Table1, “Ola” stands for olanzapine; “Clo” stands for clozapine; and “Que”stands for quetiapine, each with a point of substitution as shown:

TABLE 1 Compounds of the general formula L-M-V Com- pound No. L M V 1Ola absent —CH(NH₂)(COOCH₃) 2 Ola absent —CH(NH₂)(COOCH₂CH₃) 3 Ola—OCH₂— —CH(NH₂)(COOCH₃) 4 Ola —OCH₂— —CH(NH₂)(COOCH₂CH₃) 5 Ola absent—CH(NHCH₃)(COOH) 6 Ola absent —CH(NHCH₃)(COOCH₃) 7 Ola absent—CH(NHCH₃)(COOCH₂CH₃) 8 Ola absent —C(NH₂)(CH₂OH)(COOH) 9 Ola absent—C(NH₂)(CH₂OH)(COOCH₃) 10 Ola absent —C(NH₂)(CH₂OH)(COOCH₂CH₃) 11 Ola—OCH₂— —CH(NHCH₃)(COOH) 12 Ola —OCH₂— —CH(NHCH₃)(COOCH₃) 13 Ola —OCH₂——CH(NHCH₃)(COOCH₂CH₃) 14 Ola —CH₂CH₂OCH₂— —CH(NH₂)(COOCH₃) 15 Ola—CH₂CH₂OCH₂— —CH(NH₂)(COOCH₂CH₃) 16 Ola —CH₂CH₂OCH₂— —CH(NHCH₃)(COOCH₃)17 Ola —CH₂CH₂OCH₂— —CH(NHCH₃)(COOCH₂CH₃) 18 Clo absent —CH(NH₂)(COOCH₃)19 Clo absent —CH(NH₂)(COOCH₂CH₃) 20 Clo —OCH₂— —CH(NH₂)(COOCH₃) 21 Clo—OCH₂— —CH(NH₂)(COOCH₂CH₃) 22 Clo absent —CH(NHCH₃)(COOH) 23 Clo absent—CH(NHCH₃)(COOCH₃) 24 Clo absent —CH(NHCH₃)(COOCH₂CH₃) 25 Clo absent—C(NH₂)(CH₂OH)(COOH) 26 Clo absent —C(NH₂)(CH₂OH)(COOCH₃) 27 Clo absent—C(NH₂)(CH₂OH)(COOCH₂CH₃) 28 Clo —OCH₂— —CH(NHCH₃)(COOH) 29 Clo —OCH₂——CH(NHCH₃)(COOCH₃) 30 Clo —OCH₂— —CH(NHCH₃)(COOCH₂CH₃) 31 Clo—CH₂CH₂OCH₂— —CH(NH₂)(COOCH₃) 32 Clo —CH₂CH₂OCH₂— —CH(NH₂)(COOCH₂CH₃) 33Clo —CH₂CH₂OCH₂— —CH(NHCH₃)(COOCH₃) 34 Clo —CH₂CH₂OCH₂——CH(NHCH₃)(COOCH₂CH₃) 35 Que absent —CH(NH₂)(COOCH₃) 36 Que absent—CH(NH₂)(COOCH₂CH₃) 37 Que —CH₂— —CH(NH₂)(COOCH₃) 38 Que —CH₂——CH(NH₂)(COOCH₂CH₃) 39 Que absent —CH(NHCH₃)(COOH) 40 Que absent—CH(NHCH₃)(COOCH₃) 41 Que absent —CH(NHCH₃)(COOCH₂CH₃) 42 Que absent—C(NH₂)(CH₂OH)(COOH) 43 Que absent —C(NH₂)(CH₂OH)(COOCH₃) 44 Que absent—C(NH₂)(CH₂OH)(COOCH₂CH₃) 45 Que —CH₂— —CH(NHCH₃)(COOH) 46 Que —CH₂——CH(NHCH₃)(COOCH₃) 47 Que —CH₂— —CH(NHCH₃)(COOCH₂CH₃) 48 Que —CH₂——C(NH₂)(CH₂OH)(COOH) 49 Que —CH₂— —C(NH₂)(CH₂OH)(COOCH₃) 50 Que —CH₂——C(NH₂)(CH₂OH)(COOCH₂CH₃) 51 Que —CH₂CH₂OCH₂— —CH(NH₂)(COOCH₃) 52 Que—CH₂CH₂OCH₂— —CH(NH₂)(COOCH₂CH₃) 53 Que —CH₂CH₂OCH₂— —CH(NHCH₃)(COOCH₃)54 Que —CH₂CH₂OCH₂— —CH(NHCH₃)(COOCH₂CH₃)

The present invention thus also provides any one of Compounds 1 through54 listed in Table 1, a salt thereof, a prodrug thereof, and astereoisomer thereof.

The present invention further encompasses active compounds which arebased on mono- and bicyclic antipsychotic agents such amisulpride,sulpiride, spiperone, remoxipride, and alizapride. Such compounds may beutilized as the CNS active moiety, L, in the general formula L-M-V.

For example, the monocyclic anti-psychotic agent amisulpride may beconjugated to a linker at any one of the positions (others are alsopossible) shown with an arrow:

Similarly, alizapride, a bicyclic anti-psychotic, may be modified at theshown positions:

The compounds of the invention may be prepared following total synthesisfrom commercially available starting materials or intermediates. Asdemonstrated below, olanzapine derivatives were prepared step-wise fromsubstituted thiophene. The synthesis of clozapine proceeded step-wisefrom a substituted benzene. Once the backbone of the active moiety wasprepared, substitution with an appropriate group, constituting thelinker and/or the modulator of the glutamate NMDA receptor, waspossible.

The quetiapine derivatives were prepared via total-synthesis oralternatively via direct alkylation of the free hydroxyl group of thequetiapine skeleton.

Thus, in another aspect of the present invention, there is provided amethod for the preparation of a compound of general formula L-M-V, saidmethod comprising:

(a) providing a reactive precursor of a CNS active moiety, L;

(b) reacting said precursor under appropriate conditions with

-   -   (i) a derivative of a linker, M, and/or    -   (ii) a derivative of a the modulator of the glutamate NMDA        receptor, V, or    -   (iii) a pre-synthesized product of a linker and a modulator of        the glutamate NMDA receptor having the formula -M-V;        whereby under the reaction conditions said reactive precursor of        a CNS active moiety reacts with one of (i)-(iii) to afford,        respectively,    -   (1) a CNS active moiety substituted with a linker, M, i.e.,        having the general formula L-M; or    -   (2) a CNS active moiety substituted with a modulator of the        glutamate NMDA receptor modulator, V, i.e., having the general        formula L-V (M being absent); or a CNS active moiety substituted        with -M-V (in case steps (i) and (ii) are sequentially        followed); or    -   (3) a CNS active moiety substituted with a modulator of the        glutamate NMDA receptor modulator, V, through a linker, M, i.e.,        having the general formula L-M-V.

Where the intermediate is a precursor of L-M-V, being, for example, ofthe general structure L-M, the intermediate may be further reacted withan appropriate precursor of the modulator of the glutamate NMDA receptorto afford a compound of the general structure L-M-V.

The reactive precursor of a CNS active moiety, L, is a precursor of theCNS active moiety which may be modified, by methods known in the art, toafford a CNS active moiety conjugated to a linker. The precursor may beone having a free amine group, an alcohol, a thiol, an aldehyde, aketone, a carboxylic acid or an active carbon group, through whichconjugation may take place. The conjugation of the moiety to the linkermay take place, depending on the specific reaction conditions employed,under such experimental conditions as would be known to a person skilledin the art, to afford one or more of the following: high yield,selectivity, preference to a single isomer, etc.

The synthesis of specific isomers can be carried out employing methodswithin the knowledge of one skilled in the art, for example,stereochemically controlled synthesis using chiral synthons or chiralreagents.

The compounds of the invention typically contain at least one basic atomor substituent, and thus are capable of forming a wide variety ofdifferent salts with various inorganic and organic acids. Although suchsalts must be pharmaceutically acceptable for administration to animals(human and non-human), it is often desirable in practice to initiallyisolate the base compounds from the reaction mixture as othernon-acceptable salts, such as perchlorates, picolinates, picrates, orthe like, and then convert them to the free base compound by treatmentwith an alkaline reagent, as known to a person skilled in the art.Subsequently, the free base forms may be converted to thepharmaceutically acceptable acid addition salts.

The acid addition salts of the compounds of this invention are readilyprepared by treating the compounds with equivalent amounts of a chosenmineral or organic acid in an aqueous solvent or in a suitable organicsolvent, such as methanol or ethanol. The desired solid salt may then bereadily obtained by, e.g., evaporation of the solvent.

The pharmaceutically acceptable acid forms of the compounds of theinvention, are obtained from non-toxic acid addition salts, i.e., saltscontaining pharmaceutically acceptable anions, such as thehydrochloride, hydrobromide, hydroiodide, nitrate, sulfate or bisulfate,phosphate or acid phosphate, acetate, lactate, citrate or acid citrate,tartrate or bi-tartrate, succinate, maleate, fumarate, gluconate,saccharate, benzoate, methanesulfonate, ethanesulfonate,benzenesulfonate, and p-toluenesulfonate.

Certain compounds of the present invention have at least one acidicgroup and are thus capable of forming base salts with variouspharmaceutically acceptable cations. Examples of such salts include thealkali metal or alkaline-earth metal salts and, particularly, the sodiumand potassium salts.

The chemical bases which are used as reagents to prepare thepharmaceutically acceptable base salts of compounds of the presentinvention are those which form non-toxic base salts with the hereindescribed acidic derivatives. These particular non-toxic base saltsinclude those derived form such pharmaceutically acceptable cations assodium, potassium, calcium and magnesium, etc. These salts can easily beprepared by treating the compounds of the invention having at least oneacidic group with an aqueous solution containing the desiredpharmaceutically acceptable cation, and then evaporating the resultingsolution to dryness, in some embodiments under reduced pressure.Typically, stoichiometric quantities of reagents are preferably employedin order to ensure completeness of reaction and maximum production ofyields of the desired final product.

Compounds of the invention having both acidic and basic groups may alsobe obtained as internal salts (Zwitter ions).

The present invention also relates to prodrug derivatives of thecompounds of the invention. As known to the person skilled in the art,the term “prodrug” refers to pharmacologically inactive precursors of adrug that may be converted into its therapeutically active form underphysiological conditions in vivo, for example, when they undergosolvolysis, or enzymatic degradation in blood, or in cells, (See asbackground reference: The Organic Chemistry of Drug Design and DrugAction, Academic Press, San Diego, Calif., 1992).

Within the scope of the invention, the term also encompasses anycovalently bonded carriers, which release the active compound in vivowhen administered to an animal. Prodrug modifications of a compoundoften offer advantages of solubility, bioavailability, absorption,tissue compatibility, tissue distribution, or delayed release in themammalian organism. While the prodrug derivatives of compounds of theinvention have groups cleavable under metabolic conditions, for example,pharmaceutically acceptable esters, or amides, it is to be understoodthat such cleaving does not refer to cleaving of a bond between moietiesL and M and/or V and M in the general formula L-M-V. The cleavablegroups being different from L, M and V can be cleaved enzymatically ornon-enzymatically, or hydrolytically to the free hydroxy, carboy, oramino group of the active parent compound.

The prodrug may also be a reduced form, which is oxidized in vivo to thetherapeutic compound, for example, an alcohol to a carboxylic acid.

Thus, the present invention provides compounds, salts thereof (beingpharmaceutically acceptable or unacceptable), internal salts thereof,hydrates thereof, polymorphs thereof, prodrugs thereof and mixtures ofany one form thereof.

Pure compounds may be obtained following methods of purification asknown in the art. Where the reaction product is a mixture of isomers,specific isomers may be separated by means of classical separationtechniques, such as chromatographic or crystallization methods, or byother methods known in the art, such as through formation ofdiastereomeric salts, for example by salt formation with anenantiomerically pure chiral acid, or by means of chromatography, forexample by using chromatographic materials modified with chiral ligands.

The compounds of the invention, as shown herein are modulators of theglutamate NMDA receptor. Within the scope of the present application,the term “modulator” refers to the ability of compounds of the inventionto affect (alter) the activity of the glutamate NMDA receptor. Thereceptor may be over or under activated in response to treatment withone or more of the compounds of the invention. The over- orunder-activation of the receptor may be determined by e.g., a functionalassay or other in vitro, in vivo, and/or ex-vivo tests such as thosedemonstrated hereinbelow or such as those known to a person skilled inthe art. Thus, in some embodiments, the compounds of the invention areagonists, namely having the ability to activate the glutamate NMDAreceptor, or partial agonists, namely only partially activating thereceptor. In some other embodiments, the compounds are antagonists,namely having the ability to block or arrest the activity of theglutamate NMDA receptor, or partial antagonist.

In another aspect, the present invention provides the use of at leastone compound (or a salt, prodrug, or a stereoisomer thereof) accordingto the invention for the preparation of a composition. In someembodiments, the composition of the invention is a pharmaceuticalcomposition, comprising also at least one pharmaceutically acceptablecarrier, diluent or excipient.

The pharmaceutical composition of the invention may comprise one or morecompounds according to the invention. Where the composition comprisestwo or more compounds, the compounds may be compounds of differentclasses (e.g., one compound being a tricyclic psychotropic and the otheran antidepressant), the compounds may be salt forms of the same compound(e.g., one compound being a sodium salt of Compound 1 and the other apotassium salt of Compound 1), different compounds in different or sameform (e.g., one may be a salt and the other may be an ester), indifferent or same concentrations, etc.

The choice of carrier will be determined in part by the particularcompound, as well as by the particular method used to administer thecomposition comprising it. Accordingly, there is a wide variety ofsuitable formulations of the pharmaceutical composition of the presentinvention. The following formulations for oral, aerosol, parenteral,subcutaneous, intravenous, intramuscular, interperitoneal, rectal, andvaginal administration are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions.

Liquid formulations may include diluents, such as water and alcohols,for example, ethanol, benzyl alcohol, and the polyethylene alcohols,either with or without the addition of a pharmaceutically acceptablesurfactant, suspending agent, or emulsifying agent.

Capsule forms can be of the ordinary hard- or soft-shelled gelatin typecontaining, for example, surfactants, lubricants, and inert fillers,such as lactose, sucrose, calcium phosphate, and cornstarch.

Tablet forms can include one or more of lactose, sucrose, mannitol, cornstarch, potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodiumtalc, magnesium stearate, calcium stearate, zinc stearate, stearic acid,and other excipients, colorants, diluents, buffering agents,disintegrating agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers.

Lozenge forms can comprise a compound of the invention in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to a compound of the invention, such carriers asare known in the art.

The compounds of the present invention, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound can be administered in a physiologically acceptable diluentin a pharmaceutical carrier, such as a sterile liquid or mixture ofliquids, including water, saline, aqueous dextrose and related sugarsolutions, an alcohol, such as ethanol, isopropanol, or hexadecylalcohol, glycols, such as propylene glycol or polyethylene glycol,glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers,such as polyethyleneglycol) 400, an oil, a fatty acid, a fatty acidester or glyceride, or an acetylated fatty acid glyceride with orwithout the addition of a pharmaceutically acceptable surfactant, suchas a soap or a detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid, Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters, Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyl dialkyl ammoniumhalides, and alkyl pyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxy-ethylenepolypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl-β-aminopriopionates, and 2-alkyl-imidazolinequaternary ammonium salts, and (3) mixtures thereof.

The parenteral formulations will typically contain from about 0.5 toabout 25% by weight of the compound of the invention in solution.Suitable preservatives and buffers can be used in such formulations. Inorder to minimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. Suitable surfactants include polyethylene sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The compounds of the present invention may be made into injectableformulations. The requirements for effective pharmaceutical carriers forinjectable compositions are well known to those of ordinary skill in theart. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co.,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4^(th) ed., pages 622-630(1986).

The compositions of the invention may comprise at least one compound ora pharmaceutically acceptable salt or derivative thereof and in additioncomprise a carrier. Additionally, the composition may comprise at leastone drug selected from CNS depressants, CNS stimulants, and drugs thatselectively modify CNS function, such as anticonvulsants,anti-Parkinsonian drugs, opioid and non-opioid analgesics, appetitesuppressants, antiemetics, analgesic-antipyretics, certain stimulants,antidepressants, antimanic agents, antipsychotic agents, sedatives andhypnotics.

The pharmaceutical compositions of the invention may be administered toa subject, a human or non-human, via any of the administration routesdisclosed hereinbefore. The pharmaceutical compositions may be used in atreatment regime of a disease or disorder of choice, as known to amedical practitioner. In some embodiments, the disease or disorder is apsychological or psychiatric disease or disorder.

Thus, the present invention also provides a use of a compound accordingto the invention in the treatment of a disease or disorder.

The invention further provides a method of treating a disease ordisorder comprising administering to a subject in need thereof acompound or a composition according to the present invention.

In some embodiments, said disease or disorder is a psychiatric diseaseor disorder. In some other embodiments, said disease or disorder isassociated with modulation of the activity of the glutamate NMDAreceptor. Non-limiting examples of such diseases and disorders arediseases and disorders of the CNS, such as psychotic disorders, anxietydisorders, dissociative disorder, personality disorders, mood disorders,effective disorder, neurodegenerative disorders, convulsive disorders,boarder line disorders and mental diseases and disorders.

In other embodiments, the disease or disorder is selected fromSchizophrenia, bipolar disorders (and maintenance for bipolarity),psychotic depression, delusional disorders, conduct disorders,psychosis-induced dementia, organic psychosis, mood disorders, Torte'ssyndrome, depression, post-traumatic stress disorder, anxiety, panicdisorder and Alzheimer's disease.

The invention further provides a method of modulating the activity ofthe glutamate NMDA receptor, said method comprising contacting a tissue(e.g., a CNS tissue) expressing said glutamate NMDA receptor with atleast one compound or a composition according to the invention. Thetissue being contacted with the at least one compound or compositionaccording to the invention may be a tissue extracted or removed from thebody of the animal (ex viva) or a tissue in the body of the animal (invivo).

In some embodiments, said activity is enhanced. In some otherembodiments, said activity is decreased.

Also provided by the present invention is a method for modulating one ormore biological and/or pharmacological pathways, whereby said modulationensues the treatment of an at least one psychological and/or psychiatricdisease or disorder or the preventing of such a disease or disorder,said method comprising administering to a subject an effective amount ofa compound or a composition according to the present invention.

As used herein, the term “effective amount” refers to an amount of acompound of the invention, or a composition comprising thereof which iseffective in treating at least one disease or disorder as defined. Theamount must be effective to achieve the desired therapeutic effect asdescribed, i.e., of the activity of the glutamate NMDA receptor,depending, inter alia, on the type and severity of the disease to betreated and the treatment regime. The effective amount is typicallydetermined in appropriately designed clinical trials (dose rangestudies) and the person versed in the art will know how to properlyconduct such trials in order to determine the effective amount. Asgenerally known, an effective amount depends on a variety of factorsincluding the affinity of the ligand to the receptor, its distributionprofile within the body, a variety of pharmacological parameters such ashalf-life in the body, on undesired side effects, if any, on factorssuch as age and gender, etc.

The term “treatment” or any lingual variation thereof, as used herein,refers to the administering of a therapeutic amount of the compound ofthe invention or a composition comprising thereof which is effective toameliorate undesired symptoms associated with a disease, to prevent themanifestation of such symptoms before they occur, to slow down theprogression of the disease, slow down the deterioration of symptoms, toenhance the onset of remission period, slow down the irreversible damagecaused in the progressive chronic stage of the disease, to delay theonset of said progressive stage, to lessen the severity or cure thedisease, to improve survival rate or more rapid recovery, or to preventthe disease form occurring or a combination of two or more of the above.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, embodiments will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIGS. 1A-D show the open filed results following i.p. administration ofCompound 1 at 12.5, 25, 50, 75 and 100 mg/kg (each point is themean+/−SEM of 3-5 determinations). FIG. 1A shows the effect on distancemoved; FIG. 1B shows the effect ion velocity; FIG. 1C shows the effecton rearing frequency; and FIG. 1D shows the duration of the “not moving”periods.

FIGS. 2A-D show (each point is the mean+/−SEM of 6 determinations) thedose-dependent effect of Compound 6 (PGW-5) on the motility of mice.FIG. 2A shows the effect on distance moved; FIG. 2B shows the effect onvelocity mean; FIG. 2C shows the effect on rearing frequency; and FIG.2D shows the total duration of the “not moving” periods.

FIGS. 3A-C show the effect of oral administration of Compound 1 on thehorizontal and vertical behavior of mice. FIG. 3A shows the effect onthe distance moved; FIG. 3B shows the effect on velocity; and FIG. 3Cshows the effect on number of rearings.

FIGS. 4A-C show the effect of Compound 3 (PGW-7) (12.5, 25 and 50 mg/kg,each point is the mean+/−SEM of 6 determinations) in the open filedtest. FIG. 4A shows the effect on the distance moved; FIG. 4B shows theeffect on the total duration of “not moving” periods and FIG. 4C showsthe effect on the rearing frequency.

FIG. 5 shows the effect of Compound 1, olanzapine and D-serine (20mg/kg, p.o) on time spent in the center of an open filed and onfrequency to the center.

FIGS. 6A-B show the effect of Compound 1 (20 mg/kg, p.o.) onamphetamine-induced hyperactivity as manifested by the total number ofrearings and climbings over a period of 1 hr (FIG. 6A) and the effect ofCompound 1 (20 mg/kg, p.o.) on number of head movements (FIG. 6B).

FIGS. 7A-B show the effect of Compound 3 (PGW-7) (12.5, 25, 50 mg/kg,i.p.), amphetamine (2 mg/kg, i.p.) and a combination thereof in the Openfield test on distance moved (FIG. 7A) and immobility total duration(FIG. 7B).

FIGS. 8A-E show the effect of Compound 1 (PGW), MK-801 and a combinationthereof in the open field test on the distance moved (FIG. 8A); velocitymean (FIG. 8B); strong mobility total duration(s) (FIG. 8C); “notmoving” total duration (FIG. 8D); and rearing frequency (FIG. 9E).

FIGS. 9A-D show the effect of Compound 1 (PGW-4) (administered i.p.) oninhibition of the hyperlocomotion effect of MK-801. FIG. 9A shows thedistance moved total; FIG. 9B shows the velocity mean; FIG. 9C shows theeffect of Compound 1, MK-801 and a combination thereof on in zonefrequency; and FIG. 9D shows the effect of Compound 1, MK-801 and acombination thereof on in zone total duration.

FIGS. 10A-D shows the effect of Compound 3 (PGW-7) (12.5, 25 and 50mg/kg, i.p.) on hyperactivity-induced by MK-801 (0.15 mg/kg, i.p.). FIG.10A shows effect of Compound 3, MK-801, and a combination thereof ondistance moved; FIG. 10B shows the effect of Compound 3, MK-801, and acombination thereof on “not moving” total duration; FIG. 10C shows theeffect of Compound 3, MK-801, and a combination thereof on in-zonefrequency (zone 3); and FIG. 10D shows the effect of Compound 3, MK-801,and a combination thereof on in-zone total duration.

FIG. 11 shows the effect of Compound 1 (PGW) (30, 40 and 50 mg/kg, i.p.)on catalepsy.

FIGS. 12A-D show the effect of Compound 1 (POW) (10, 20 mg/kg, oral), inthe forced swim test (FST) on distance moved (FIG. 12A); on velocity(FIG. 12B); on strong mobility (FIG. 12C); and on immobility (FIG. 12D).

FIGS. 13A-C show the effect of Compound 1 (PGW-4) (10, 20, 30 mg/kg,oral) in the FST on immobility (FIG. 13A); on distance moved (FIG. 13B);and on strong mobility (FIG. 13C).

FIGS. 14A-B show the effect of olanzapine in comparison to Compound 1(POW) in the FST on distance moved (FIG. 14A) and immobility (FIG. 14B).

FIGS. 15A-E show the effect of Compound 1 (PGW-4) and diazepam in theelevated plus maze test on mice frequency in-zone (FIG. 15A); on totalduration in zone (FIG. 15B); on velocity (FIG. 15C); and rearingfrequency (FIG. 15D). FIG. 15E shows the effect of Compound 6 (PGW-5) inthe elevated plus maze test.

FIG. 16 shows the effect of 10 and 20 mg/kg of Compound 1 (PGW-4),administered p.o. on latency to platform.

FIG. 17 shows the effect of acute treatment of Compound 1 at 10 and 20mg/kg given p.o. on the spatial cognitive tasks in the Morris Water Mazein rats pretreated with MK-801.

FIG. 18 shows the effect of acute use of Compound 6 at 10 mg/kg givenp.o. on the spatial cognitive tasks in the Morris Water Maze in ratspretreated with MK-801.

FIG. 19 shows the effect of Compound 1 on the mice body weight.

FIG. 20 shows the effect of treatments with different drugs on gliomaviability.

DETAILED DESCRIPTION OF THE INVENTION A. Synthesis of Novel Compounds

The compounds of the invention may be prepared according to a variety ofprocedures, as disclosed and exemplified herein. One general way toprepare the compounds of the general formula (A), substituted with theappropriate amino acid, e.g., glycinyl, serinyl or sarcosinyl moietiesdirectly or through a linker, as shown in the specific structuresherein, is to first prepare the demethylated derivatives of the parentCNS-active agents (e.g. des-Me-olanzapine, des-Me-clozapine). These canbe prepared by coupling two ortho-disubstituted aromatics: one bearing anitro group in an ortho position to a good leaving group (e.g.,fluoride, chloride), while another is an ortho-cyano aromatic amine,phenol or thiophenol. The coupling (for example through an aromaticnucleophilic substitution) forms a nitro cyano-diarylamine, nitrocyano-diarylether, or nitro cyano-diarylthioether. In the next stage,the nitro group is reduced to amine with a simultaneous centraldiazepine ring closure. The formed tricyclic anticline reacts withpiperazine, forming the des-Me form of the L fragment of the targetcompound, as disclosed and exemplified herein.

The piperazine secondary amine of this intermediate can be furtherreacted with a β-iodo-alanine, or dehydroalanine derivative (where thelinker moiety is absent) or with O-iodoalkyl serine derivatives (wherethe linker moiety is present). The amine of the aminoacid moiety may beprotected utilizing any protecting groups known to the person skilled inthe art. Typically, in the following examples, the amine was protectedwith the Boc-protecting group. Deprotection at the final step with anacid such as HCl, afforded the desired active compound in thetrihydrochloride salt form.

It should be noted that the following examples are non-limiting innature and are presented for the full understanding of the invention.The synthetic procedures provided may be varied mutatis mutandis andother novel compounds of the general formulas disclosed herein may beprepared.

Synthesis of Compound 1 (1) Synthesis of2-Amino-5-methyl-3-thiophenecarbonitrile (A)

For a general synthesis see He, X.; Griesser, U. J.; Stowell, J. G.;Borchrdt, T. B.; Byrn, S. R. J. Pharm. Sci. 2001, 90, 371.

Sulfur (S₈, 0.9 g, 0.0 27 mmol), propionalaldehyde (2 ml, 0.027 mmol)and DMF (6 ml) were transferred to a three-necked round-bottomed flaskequipped with a dropping funnel and condenser. The resulting mixture wascooled to 0° C., and triethylamine (2.3 ml) was added in a dropwisemanner via the dropping funnel. The resulting dark solution was thenwarmed to room temperature over a period of 1 h. A solution ofmalononitrile (1.71 ml, 1.8 g, 0.027 mmol) in DMF (3.2 ml) wastransferred to the addition funnel and added in a dropwise manner. Theresulting brownish mixture was stirred overnight at room temperature.Then the mixture was poured over 80 ml of ice and water to yield anorange precipitate. The solid A was filtered, washed with chilled water,and dried in vacuo; yield 78%.

¹H NMR (200 MHz, CDCl₃): δ 6.35 (s, 1H), 4.15 (br s, 2H), 2.27 (s, 3H).¹³C NMR (50 MHz, CDCl₃): δ 160.9, 146.3, 124.6, 122.0, 115.7, 14.9.

(2) Synthesis of 2-(2-nitroanilido)-5-methyl-3-thiophenecarbonitrile (B)

For a general synthesis see He, X.; Griesser, U. J.; Stowell, J. G.;Borchrdt, T. B.; Byrn, S. R. J. Pharm. Sci. 2001, 90, 371.

To NaH (3 equivalents, from 55% suspension in oil, rendered oil-free bywashing with hexane) was added 1 ml of dry THF.2-amino-5-methyl-3-thiophenecarbonitrile (A, as prepared above, 0.5 g,3.6 mmol) and 4-fluoro-3-nitotoluene (0.51 g, 3.6 mmol) were dissolvedin dry THF (1.5 ml) and added in a dropwise manner to the suspensionwhile the temperature was maintained below 30° C. The reaction mixturewas allowed to stir overnight under N₂ purge. The mixture was thenpoured into 11 ml of ice-water mixture, neutralized with concentratedHCl, and extracted with 36 ml of DCM. The DCM solution was dried overMgSO₄ and evaporated to dryness. The residue was purified by flashchromatography on silica gel (elution with 1:9 EtOAc/Hexanes) to givecompound B (yield 60%).

¹H NMR (200 MHz, CDCl₃): δ 9.61 (br s, 1H), 8.25 (dd, J=8.5 Hz, J=1.5Hz, 1H), 7.52 (dt, J=7.8 Hz, J=1.4 Hz, 1H), 7.19 (dd, J=8.5 Hz, J=1.1Hz, 1H), 6.97 (dt, J=7.8 Hz, J=1.2 Hz, 1H), 6.78 (d, J=1.1 Hz, 1H), 2.48(s, 3H).

(3) Synthesis of 4-amino-2-methyl-10H-thieno[2,3-b][1,5]benzodiazepine,hydrochloride salt (C)

For a general synthesis see Chakrabarti, J. K.; Hotten, T. M.; Pullar,I. A.; Steggles, D. J. J. Med. Chem. 1989, 32, 2375.

To a slurry of B (100 mg, 0.4 mmol) in ethanol (1 ml) was addedtin(II)-chloride-dihydrate (260 mg, 1.16 mmol) in concentrated HCl (1ml), the solution was heated to reflux for 3 h and cooled overnight, andthe solid C was filtered, washed with chilled DCM, and dried in vacuo;yield 95%.

¹H NMR (200 MHz, DMSO-d₆): δ 11.05 (s, 1H), 9.53 (s, 1H), 9.08 (br s,1H), 8.82 (br s, 1H), 6.80-7.14 (m, 4H), 6.78 (s, 1H), 2.23 (s, 3H).

(4) Synthesis of 2-methyl-4-(1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine (D)

For a general synthesis, see Chakrabarti, J. K.; Hotten, T. M.; Pullar,I. A.; Steggles, D. J. J. Med. Chem. 1989, 32, 2375.

Starting material C (prepared according to the above, 140 mg, 0.53 mmol)was added to a mixture of dry dimethyl sulfoxide (1 ml), dry toluene (1ml), and piperazine (140 mg, 1.6 mmol). The stirred solution was thenheated at 125° C. under nitrogen for 5 h and cooled to room temperature.Then distilled water (2 ml) was added, while the temperature is keptbelow 25° C. After stirring at 5° C. for 30 minute, the suspension wascooled overnight and the solid D was filtered, washed with chilledwater, and dried at 70° C. under reduced pressure; yield 69%.

¹H NMR (200 MHz, CDCl₃): δ 6.80-7.06 (m, 3H), 6.60 (dd, J=7.4 Hz, J=1.3Hz, 1H), 6.30 (d, J=0.8 Hz, 1H), 5.01 (s, 1H), 3.48 (m, 4H), 2.95 (m,4H), 2.31 (s, 3H).

(5) Synthesis of2-tert-butoxycarbonylamino-3-[4-(2-methyl-10H-thieno[2,3-b][1,5]benzodiazepine-4-yl)-piperazin-1-yl]-propionicacid, methyl ester (E)

A mixture of D (200 mg, 0.67 mmol), Boc-iodo-Ala-OMe (220 mg, 0.67 mmol)and Na₂CO₃ (71 mg, 0.67 mmol) in dry acetone (17 ml) was refluxedovernight under nitrogen. The solvent was evaporated, the residuediluted with dry methanol (2 ml, distilled from magnesium), and thereaction mixture was stirred under N₂ overnight at 45° C. Methanol wasevaporated. Chromatography on silica gel afforded E by elution with 1:1EtOAc:hexanes; yield 48%.

¹H NMR (400 MHz, CDCl₃): δ 7.03 (d, J=7.6 Hz, 1H), 6.95 (t, J=7.6 Hz,1H), 6.87 (t, J=7.6 Hz, 1H), 6.61 (d, J=7.5 Hz, 1H), 6.26 (s, 1H), 5.32(br s, 1H), 4.35 (br m, 1H), 3.74 (s, 3H), 3.49 (br s, 4H), 2.73 (br m,2H), 2.53 (br in, 4H), 2.30 (s, 31-1), 1.45 (s, 9H). ¹³C NMR (100 MHz,CDCl₃): δ 172.6, 157.7, 155.5, 152.4, 142.9, 140.1, 129.1, 128.0, 124.6,124.0, 122.8, 119.1, 80.0, 58.8, 53.2, 52.3, 51.9, 47.0, 28.3, 15.4.

(6) Synthesis of2-amino-3-[4-(2-methyl-10H-thieno[2,3-b][1,5]benzodiazepin-4-yl)-piperazin-1-yl]-propionicacid, methyl ester hydrochloride salt (Compound 1-salt)

A mixture of E (80 mg, 0.16 mmol) and HCl/methanol (1.25 M, 2 ml) wasstirred overnight. After evaporating the solvent, the residue wasdiluted with water and washed with ether. The aqueous solution wasevaporated to dryness to give the protonated Compound 1; yield 92%.

¹H NMR (400 MHz, D₂O): δ 6.99 (dt, J=7.4 Hz, J=1.3 Hz, 1H), 6.83-6.92(m, 2H), 6.63 (d, J=7.9 Hz, 1H), 6.16 (s, 1H), 4.50 (m, 1H), 3.78 (br s,4H), 3.66 (s, 3H), 3.58 (dd, =14.3 Hz, J=6.2 Hz, 1H), 3.34 (dd, J=14.3Hz, J=6.3 Hz, 1H), 3.28 (br s, 4H), 2.02 (s, 3H). ¹³C NMR (100 MHz,D₂O): δ 167.4, 164.2, 160.5, 147.1, 132.3, 129.3, 127.2, 125.7, 125.1,121.9, 120.1, 109.7, 55.1, 54.4, 52.1, 48.3, 47.5, 14.3. MS (FAB):calcd. for C₂₀H₂₆N₅O₂S (MH⁺) 400.1. found 400.1.

Synthesis of Compound 2 (1) Synthesis of N-Boc-L-serine, ethyl ester (F)

To a solution of Boc-Ser-OH (0.3 g, 1.46 mmol, 1 eq) in dry DMF (4 ml)at 0° C., dry K₂CO₃ (0.22 g, 1.6 mmol, 1.1 eq) was added. The resultingwhite suspension was stirred for 15 minute and ethyl iodide (0.68 g,0.35 ml, 4.38 mmol, 3 eq) was dropwise added. The cooling was removedand reaction mixture was stirred at room temperature under N₂ for 24 h.To the formed white emulsion H₂O was added, and the mixture extracted byEtOAc. The combined organic layer was washed by H₂O and then brine, anddried over MgSO₄. The solvent was evaporated and the obtained oil wasdried overnight in high vacuum. Yield of the pure F—0.27 g (79%).

¹H NMR (400 MHz, CDCl₃): δ 5.55 (d, 1H), 4.27 (m, 1H), 4.16 (q, J=8.0Hz, J=6.2 Hz, 2H), 3.79-3.90 (m, 2H), 3.24 (br 5, 1H), 1.39 (s, 9H) 1.25(t, J=14.2 Hz, 3H).

(2) Synthesis of N-Boc-dehydroalanine, ethyl ester (G)

To a solution of Boc-L-serine ethyl ester, F, as prepared above, (0.45g, 1.9 mmol, 1 eq) in dry DCM (10 ml) under N₂, triethylamine (0.3 ml,2.1 mmol, 1.1 eq) was added and then dichloro-acetylchloride (0.2 ml,2.1 mmol, 1.1 eq) was added dropwise. The reaction mixture was stirredat room temperature for 1 h and then the solution of DBU (0.31 ml, 2.1mmol, 1.1 eq) in 2.5 ml of dry DCM was added. The dark blue solution wasrefluxed overnight under N₂. The cooled reaction mixture was poured to5% citric acid solution in H₂O, extracted by DCM and the combinedorganic layer was washed by brine, dried over MgSO₄ and evaporated. Theblack oil residue was dried in high vacuum and yielded 0.5 g ofintermediate dicholroacetyl derivative. This product was dissolved in 3ml of dry DCM, then DBU (0.33 ml) in 2 ml DCM was added and reactionmixture was refluxed under N₂ overnight. The cooled solution was pouredto 5% aqueous citric acid solution and extracted by DCM. The organiclayer was washed with brine, dried over MgSO₄ and evaporated. Theresulting black oil was purified by flash chromatography on silica gel(eluent—3% EtOAc in Hexanes).

Yield of the colorless oil product—0.23 g (58%)

¹H NMR (200 MHz, CDDl₃): δ 7.0 (s, 1H), 6.1 (s, 1H) 5.69 (s, 1H), 4.24(q, J=14.2 Hz, 2H) 1.45 (s, 9H), 1.29 (t, J=7.1 Hz, 2H).

(3) Synthesis of2-(N-Boc)amino-3-[4-(2-methyl-10H-thieno[2,3-b][1,5]benzodiadizepin-4-yl)-piperazin-1-yl]-propionic acid, ethyl ester (H)

A mixture of2-methyl-4-(1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine,compound D above, (0.14 g, 0.46 mmol, 1 eq) and N-Boc-dehydroalanineethyl ester (G, 0.1 g, 0.46 mmol, 1 eq) in 4 ml of anhydrous EtOH wasrefluxed at 60-65° C. overnight. The solvent was evaporated to drynessand the crude product was purified by flash chromatography (eluent—40%EtOAc in Hexanes). Yield—0.15 g (65%)

¹H NMR (400 MHz, CDCl₃): δ 6.95-7.01 (m, 2H); 6.86 (m, 1H); 6.60 (d,J=7.9 Hz, 1H); 6.26 (s, 1H); 5.35 (br s, 1H); 4.33 (br s, 1H); 4.16-4.20(m, 2H); 3.46 (br s, 4H); 2.73 (br s, 2H); 2.53-2.58 (m, 4H); 2.22 (s,3H); 1.45 (s, 9H); 1.29 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ172.9, 158.4, 156.2, 152.6, 143.3, 141.6, 129.9, 128.9, 125.4, 124.6,123.7, 120.2, 119.7, 80.7, 62.1, 59.7, 54.1, 52.8, 47.6, 29.1, 16.2,14.9.

(4) Synthesis of 2-Amino-3-[4-(2-methyl-10H-thieno[2,3-b][1,5]-benzodiazepine-4-yl)piperazin-1-yl]propionic acid, ethyl estertrihydrochloride salt (Compound 2)

A mixture of 2-(N-Boc)amino-3-[4-(2-methyl-10H-thieno[2,3-b][1,5]benzodiadizepin-4-yl)-piperazin-1-yl]-propionic acid, ethyl ester, H, (50 mg,0.096 mmole) and HCl/ethanol (1.25 M, 2 ml) was stirred overnight atroom temperature. The solvent was evaporated, the residue was dilutedwith water and washed with diethylether. The aqueous solution waslyophilized. Yield of Compound 2—30 mg (60%).

¹H NMR (400 MHz, CDCl₃): δ 7.20-7.15 (m, 1H); 7.03-7.13 (m, 2H); 6.85(d, J=8.0 Hz, 1H); 6.37 (s, 1H); 4.37 (m, 1H); 4.23 (q, J=8.0 Hz, 2H);3.78 (br s, 4H); 3.26 (dd, J=12.0 Hz, J=8.0 Hz, 1H); 3.10 (m, 2H); 2.97(br s, 4H); 2.19 (s, 3H); 1.20 (t, J=8.0 Hz, 3H). ¹³C NMR (100 MHz,CDCl₃): δ 168.8, 163.8, 160.7, 147.8, 132.9, 129.9, 128.2, 126.3, 125.9,122.9, 120.8, 111.2, 64.9, 56.2, 52.7, 50.6, 15.0, 13.8. MS (Fab):calcd. for C₂₁H₂₈N₅O₂S (MH⁺) 414.4. found 414.2.

Synthesis of Compound 6 (1) Synthesis of N-Boc-N-methyl-L-serine, methylester (I)

To a solution of Boc-N-Me-L-serine (0.5 g, 2.2 mmol, 1 eq) in dry DMF (2ml) at 0° C. dry K₂CO₃ (0.35 g, 2.5 mmol, 1.2 eq) was added. Theresulting white suspension was stirred for 15 minutes and methyl iodide(0.93 g, 0.42 ml, 6.6 mmol, 3 eq) was dropwise added. The cooling wasremoved and reaction mixture was stirred at room temperature under N₂for 24 h; the progress of the reaction was monitored by TLC(EtOAc/Hexanes 4:6). To the formed white emulsion H₂O was added, and themixture extracted by EtOAc (three times×30 ml). The combined organiclayer was washed by H₂O and then brine, dried over MgSO₄. The solventwas evaporated and the obtained colorless oil was dried overnight inhigh vacuum. Yield of the pure product I—0.4 g (76%).

Two aptamers are visible in NMR. ¹N NMR (400 MHz, CDCl₃); δ 4.47 (m,1H), 4.03 (m, 2H), 3.75-3.83 (m, 2H), 3.13 (m, 1H), 3.69 (s, 3H), 2.89(s, 3H), 2.85 (s, 3H), 1, 41 (s, 9H), 1.37 (s, 9H). ¹³C NMR (100 MHz,CDCl₃): δ 170.0, 170.6, 156.2, 154.9, 80.4, 80.2, 62.1, 61.1, 60.7,60.6, 33.7, 33.1, 28.0.

(2) Synthesis of N-Boc-N-methyl dehydroalanine, methyl ester (J)

To a solution of Boc-N-methyl-L-serine methyl ester, I, (0.46 g, 1.9mmol. 1 eq) in 10 ml dry DCM under N₂, triethylamine (0.29 ml, 2.2 mmol,1.15 eq) was added and then dichloroacetylchloride (0.23 ml, 2.2 mmol,1.15 eq) was added dropwise. The reaction mixture was stirred at roomtemperature for 1 h, then the solution of DBU (0.33 ml, 2.2 mmol, 1.15eq) in 2.5 ml dry DCM was added, and the combined mixture was refluxedovernight.

The cooled reaction mixture was poured to 5% citric acid solution inH₂O, extracted by DCM, and the separated organic layer was washed bybrine, dried by MgSO₄ and evaporated. The black oil residue (0.52 g) ofthe intermediate dichloroacetyl derivative was dissolved in 3 ml dry DCMand DBU (0.33 ml) in 2 ml DCM was added. The reaction mixture wasrefluxed under N₂ overnight. The cooled solution was poured to 5% citricacid aqueous solution, extracted by DCM, and the organic layer waswashed by brine, dried by MgSO₄ and evaporated. The crude product waspurified by flash chromatography on silica gel (elution with isocratic10% EtOAc/Hexanes) to give the pure J—yield 0.2 g (48%). Drying of theproduct in high vacuum should be avoided.

¹H NMR (200 MHz, CDCl₃): δ 5.77 (s, 1H), 5.30 (s, 1H), 3.75 (s, 3H),3.09 (s, 3H), 1.39 (s, 9H). ¹³C NMR (50 MHz, CDCl₃): δ 153.7, 141.3,115.1, 80.9, 52.0, 36.4, 27.9.

(3) Synthesis of2-(N-Boc-N-methyl)amino-3[4-(2-methyl-10H-thieno[2,3-b][1,5]-benzodiazepin-4-yl)piperazin-1-yl]propionicacid, methyl ester (K)

A mixture of2-methyl-4-(1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine,compound D above, (0.28 g, 0.93 mmol, 1 eq) and Boc-N-methyldehydroalanine methyl ester, J, (0.2 g, 0.93 mmol, 1 eq) in 5 mlanhydrous MeOH was heated at 50° C. under N₂ overnight. The cooledsolution was evaporated to dryness and the residue was purified by flashchromatography on silica gel (eluting with isocratic 50% EtOAc/Hexanes).Yield of pure K—0.3 g (63%).

¹H NMR (400 MHz, CDCl₃): δ 7.03 (d, J=7.6 Hz, 1H), 6.95 (t, J=7.6 Hz,1H), 6.87 (t, J=7.6 Hz, 1H), 6.60 (d, J=7.5 Hz, 1H), 6.29 (s, 1H), 5.02(m, 1H) 4.98 (s, 1H), 4.57 (m, 1H), 3.76 (s, 3H), 3.47 (br s, 4H), 2.91(br m, 2H), 2.84 (s, 3H), 2.68-2.90 (br m, 4H), 2.31 (s, 3H) 1.47 (s,9H). ¹³C NMR (100 MHz, CDCl₃): δ 172.15, 157.99, 155.6, 152.1, 142.9,141.3, 129.4, 128.5, 125.0, 124.1, 123.4, 113.9, 119.3, 80.8, 80.4,58.0, 57.5, 56.5, 55.7, 53.6, 53.4, 52.5, 47.3, 28.8, 15.8.

(4) Synthesis of2-N-methylamino-3-[4-(2-methyl-10H-thieno[2,3-b][1,5]-benzodiazepin-4-yl)-piperazin-1-yl]propionic acid, methyl estertrihydrochloride salt (Compound 6)

A solution of2-(N-Boc-N-methyl)amino-3[4-(2-methyl-10H-thieno[2,3-b][1,5]benzodiazepin-4-yl)piperazin-1-yl]propionicacid, methyl ester, K, (0.3 g, 0.58 mmol) in HCL/ethanol (1.25 M, 8 ml)was stirred at room temperature overnight. The light yellow precipitatewas formed and the suspension was decanted. The precipitate was washedseveral times with dry diethyl ether, and dried under vacuum.

Yield of Compound 6—0.26 g (87%).

¹H NMR (400 MHz, D₂O): δ 7.15 (dq, J=6.6 Hz, J=2.3 Hz, 1H), 7.03-7.05(m, 2H), 6.82 (d, J=7.8 Hz, 1H), 6.32 (s, 1H), 4.26 (m, 1H), 3.72 (br s,4H), 3.73 (s, 3H), 3.30 (dd, J=14.3 Hz, J=5.7 Hz, 1H), 2.97 (br s, 4H),2.74 (s, 3H), 2.17 (s, 3H). ¹³C NMR (100 MHz, D₂O): δ 167.6, 163.0,159.7, 146.9, 131.9, 128.9, 127.16, 125.4, 124.9, 122.0, 119.8, 110.1,57.0, 54.2, 53.9, 51.7, 48.6, 31.7, 14.1. MS (FAB): calcd. forC₂₁H₂₇N₅O₂S (MH⁺) 413.5. found 414.3. EA (%): calcd. for C₂₁H₃₁Cl₃N₅O₃S(M.3HCl.H₂O): C, 46.58, H, 5.91, Cl, 19.65. found: C, 45.94, H, 6.18,Cl, 19.52.

Synthesis of Compound 14 (1) Synthesis of O-Allyl-N-Boc-L-serine methylester (L)

To a stirred solution of N-Boc-L-serine methyl ester (0.22 g, 1 mmol, 1eq) in dry THF (2 ml) under N₂ a mixture of n-allylpalladium chloridedimer (0.01 g, 0.02 mmol), triphenylphosphine (0.024 g, 0.09 mmol) andallyl ethyl carbonate (0.26 ml, 2 mmol, 2 eq) in dry THF (1 ml) wasdropwise added at room temperature.

The reaction mixture was refluxed under N₂ overnight. The solvent wasevaporated and the crude product was purified by flash chromatography onsilica gel (elution with isocratic 10% EtOAc/Hexanes) to give the purecompound. Yield of L was 0.14 g (63%).

¹H NMR (400 MHz, CDCl₃): δ 5.75-5.85 (m, 1H), 5.36 (m, 1H), 5.13-5.23(m, 2H) 3.93-3.95 (m, 2H), 3.82 (dd, J=3.0 Hz, J=6.4 Hz, 1H), 3.72 (s,3H), 3.61 (dd, J=3.3 Hz, J=9.4 Hz, 1H), 1.44 (s, 9H). ¹³C NMR (100 MHz,CDCl₃): δ 171.0, 155.3, 133.8, 117.2, 79.7, 72.0, 69.7, 53.8, 52.2,28.1.

(2) Synthesis of O-(3-Iodopropyl)-N-Boc-L-serine methyl ester (M)

To a solution of O-Allyl-N-Boc-L-serine methyl ester, L prepared asabove, (0.25 g, 1 mmol, 1 eq) in dry THF (1 ml) equimolar amount of 1MBH₃-THF complex solution in THF (0.96 ml) was dropwise added at 0° C.After addition, the colorless solution was heated at 55° C. under N₂ for1.5 h. The progress of the reaction was monitored by TLC (20%EtOAc/Hexanes). To a cooled reaction mixture (0° C.) I₂ (0.17 g, 0.67mmol) was added, followed by solution of NaOH in MeOH (0.24 ml, 3M). Theformed dark solution was stirred under N₂ at room temperature for 2 h.

The reaction mixture was then poured into a 1 M solution of sodiumthiosulfate in H₂O and extracted by EtOAc (three times with 20 ml). Thecombined organic layer was washed by brine and dried over MgSO₄. Thesolvent was evaporated and the crude was purified by flashchromatography on silica gel (elution with isocratic 20% EtOAc/Hexanes)to give the pure M in 25% yield (0.093 g).

¹H NMR (400 MHz, CDCl₃): δ 5.32 (d, J=8.0 Hz, 1H), 4.4 (m, 1H), 3.81(dd, =3.2 Hz, J=9.4 Hz, 1H), 3.66 (s, 3H), 3.64 (dd, J=3.2 Hz, J=9.4 Hz,1H), 3.44-3.51 (m, 2H), 3.19 (t, J=6.6 Hz, 2H), 1.98 (quint, J=6.1 Hz,2H), 1.44 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 171.8, 156.2, 80.7, 71.6,71.3, 70.7, 54.7, 53.3, 33.7, 29.0, 3.2.

(3) Synthesis of2-(N-Boc-amino)-3-[O-propyl-3-(4-(2-methyl-10H-thieno[2,3-b][1,5]benzodiazepine-4-yl)piperazin-1-yl]propionicacid, methyl ester (N)

A mixture of2-methyl-4-(1-piperazinyl)-10H-thieno[2,3-b][1,5]benzodiazepine,(compound D, 0.073 g, 0.24 mmol, 1 eq), O-(3-Iodopropyl)-N-Boc-L-serinemethyl ester (M, 0.093 g, 0.24 mmol, 1 eq) and dry Na₂CO₃ (0.025 g 0.24mmol, 1 eq) in 8 ml anhydrous acetone was refluxed under N₂ overnight.The cooled reaction mixture was evaporated to dryness; 20 ml CHCl₃ wasadded to the residue and the precipitate of NaI was filtered off. Thesolvent was evaporated and the crude product was purified by flashchromatography on silica gel (eluting with gradient: 3% MeOH in CHCl₃ to5% MeOH in CHCl₃) to give the pure N. Yield 0.1 g (77%).

¹H NMR (400 MHz, CDCl₃): δ 7.04 (d, J=7.6 Hz, 1H), 6.97 (t, J=7.6 Hz,1H), 6.95 (t, J=7.6 Hz, 1H), 6.62 (d, J=7.6 Hz, 1H) 6.28 (d, J=0.9 Hz,1H), 5.48 (d, J=8.4 Hz, 1H), 5.01 (s, 1H), 4.42 (m, 1H), 3.84 (dd, J=9.3Hz, J=2.6 Hz, 1H), 3.75 (s, 3H), 3.63 (dd, J=9.5 Hz, J=3.2 Hz, 1H), 3.57(m, 4H), 3.47 (m, 2H), 2.55 (m, 4H), 2.46 (m, 2H), 2.30 (s, 3H), 1.77(quint, J=6.6 Hz, 2H), 1.45 (s, 9H). ¹³C NMR (100 MHz, CDCl₃): δ 172.1,158.3, 156.3, 152.5, 143.2, 141.7, 129.8, 128.8, 125.4, 124.5, 123.7,120.3, 119.7, 80.8, 71.5, 70.5, 55.9, 54.8, 53.9, 53.2, 47.5, 29.1,27.5, 16.2.

(4) Synthesis of2-Amino-3-[O-propyl-3-(4-(2-methyl-10H-thieno[2,3-b][1,5]-benzodiazepine-4-yl)piperazin-1-yl]propionicacid, methyl ester, trihydrochloride salt (Compound 14)

A mixture of2-(N-Boc-amino)-3-[O-propyl-3-(4-(2-methyl-10H-thieno[2,3-b][1,5]benzodiazepine-4-yl)-piperazin-1-yl]propionicacid, methyl ester, N, (100 mg, 0.18 mmol) and HCl/MeOH (1.25M, 3 ml)was stirred overnight at room temperature. The solvent was evaporated;the residue was diluted with water and washed with diethyl ether. Theaqueous solution was lyophilized. Yield of Compound 14: 78 mg (95%).

¹H NMR (400 MHz, D₂O): δ 7.19-7.23 (m, 1H), 7.05-7.11 (m, 2H), 6.87 (d,J=8.0 Hz, 1H), 6.41 (s, 1H), 4.30 (t, J=3.9 Hz, 1H), 3.93 (dd, J=4.3 Hz,J=11.0 Hz, 2H), 3.81 (dd, J=3.2 Hz, J=11.0 Hz, 2H), 3.76 (s, 3H), 3.75(br s, 4H), 3.57 (m, 2H), 3.26 (m, 6H), 2.22 (s, 3H), 1.98 (m, 2H). ¹³CNMR (100 MHz, D₂O): δ 169.46, 165.2, 161.7, 148.0, 133.4, 130.2, 128.0,126.4, 126.1, 122.5, 120.9, 110.8, 68.7, 68.2, 55.3, 54.5, 53.8, 51.7,47.6, 24.3, 15.0. MS (FAB): calcd for C₂₃H₃₁N₅O₃S (MH⁺) 457.5. found458.2.

B. Animal Behavioral Models

In the following tests, unless otherwise indicated, male ICR or Balb/Cmice, 6 to 8 weeks of age were used. All animals were housed (4-5/cage)under controlled conditions (temperature, light, humidity) given foodand water ad libitum and allowed for 5-7 days of acclimatization beforethe beginning of experimentation.

Compound of the invention such as Compounds 1, 3 or 6, or olanzapinewere administered orally to the mice at an equimolar dose and thebehavioral tests were performed 1-4 hr later. The following tests wereperformed:

(a) A forced swim test,

(b) An open field exploration, and

(c) An amphetamine-induced hyperactivity.

The forced swim test and the open field exploration tests weredocumented by a digital video camera linked to behavioral analysissoftware (Noldus Infoiuiation Technology, Netherlands).

Forced Swim Test (FST)

This is one of the most widely used tools for screening antidepressantactivity pre-clinically in acute treatment. The test was first describedby Porsolt et al., (Behavioral despair in mice: a primary screening testfor antidepressants. Arch. Int. Pharinacodyn. Ther. 229, pp. 327-336,1977). The FST test is based on the observation that rats and micedevelop an immobile posture when placed in an inescapable cylinder ofwater. This behavior is considered a behavioral despair as opposed toactive form of coping with stressful conditions. The FST test isconsidered a good screening tool with good reliability and predictivevalidity.

Three parameters were defined in the evaluation of FST:

1. Immobility—defined in the traditional Porsolt test as when noadditional activity is observed other than that required to keep theanimal's head above water;

2. Swimming behavior—being the movement (usually horizontal) throughoutthe chamber that also includes crossing into another quadrant;

3. Climbing behavior—being defined as the upward-directed movements(vertical) of the forepaws along the side of the swim chamber.

The test in mice was conducted acutely (60-90 minutes post oral drugadministration) and animals were dropped to the cylinder for 6 minutesand scoring was performed in the last 4 minutes after 2 minutes ofadaptation.

Round glass cylinders, 18 cm in diameter and 20 cm deep were used. Watertemperature was 24-28° C. Four parameters were taken: immobility,velocity, distance and strong mobility. The immobility in the animalswas defined by activity lower than 10% movement of the center of gravityof the animal. Swimming was defined by the distance and the velocity ofthe animal, and climbing was related to strong mobility (movement ofcenter of gravidance more than 30%).

Open Field Exploratory Locomotor Activity

The procedure consists of subjecting an animal, usually a rodent, to anunknown large environment from which escape is prevented by surroundingwalls. The open field test is now one of the most popular procedures inanimal psychology (Crawley, et al., Exploratory behavior models ofanxiety in mice, Neurosci Biobehav Rev 9 (1985), pp. 37-44). Theprocedure involves forced confrontation of a rodent with the situation.The animal is placed in the center or close to the walls of theapparatus and the following behavioral items are recorded for a periodranging from 5 to 20 minutes: horizontal locomotion, and frequency ofrearing or leaning. In such a situation, rodents spontaneously preferthe periphery of the apparatus to activity in the central parts of theopen field. Increase in time spent in the central part as well as of theratio central/total locomotion or decrease of the latency to enter thecentral part are indications of anxiolysis.

In a typical test, an individual mouse was placed in a novel environmentof a square open field (50×50 cm), the floor of which had been dividedinto 3 areas, as shown below. The area within 10 cm of the chamber wallswas termed the periphery. Animals were treated with a compound of theinvention such as Compound 1, 3 or 6 with olanzapine or with a vehicle.One hour after drug administration, the animal was placed in the samecorner of the field. The animal behavior in the open field was recordedby videotaping for 20-60 minutes and analyzed subsequently digitallyusing the Noldus software for animal behavior. The measurements includedvelocity, distance moved, frequency of visits to the central area,number of rearing events.

Amphetamine-induced hyperactivity

The amphetamine-induced hyperactivity and motility is one of the mostpopular animal models for schizophrenia (Pouzet B, et al., Effects ofthe 5-HT(7) receptor antagonist SB-258741 in animal models forschizophrenia. Pharmacol Biochem Behav April; 71(4):655-665, 2002 andGeyer, et al., Animal behavior models of the mechanisms underlyingantipsychotic atypicalicity, Progress in Neuro-Psychopharmacol &Biological Psychiatry, 27, 1071-79, 2003). Amphetamines are known toaugment dopaminergic and noradrenergic neurotransmission by inducingcatecholamine release and preventing catecholamine reuptake

The experiment was conducted with ICR male mice placed in individualbarrels. Olanzapine, or Compound 1 (for example at a dose of 10 and 20mg/kg) were administered interperitoneal (i.p.) to the mice 0.5 hourprior to subcutaneous (s.c.) administration of amphetamine (3 mg/kg).The locomotor activity, number of rearings and head movements wasrecorded every 15 minutes for 2 h.

MK-801-induced stereotype behavior in rats (in open field)-MK-801 is ananalogue of PCP which inhibits NMDA receptors and elicitshyperlocomotion and stereotypic behavior in rodents and is used as ananimal model of schizophrenia (Stephen, et al., Topiramate antagonizesMK-801 in an animal model of schizophrenia, Eur. J. Pharmacol. 449,2002, 121-5). For experimentation, naïve Balb/c mice were used. MK-801(0.15 mg/kg i.p) was administered 40 minutes after Compound 1 (or anyone of the other compounds of the invention) or a vehicle, and 20minutes prior to placing the mouse in an open field.

Animals placed in the open field were followed for 60 minutes and theirdistance moved, velocity, duration of immobility, and number of rearingevents were registered using the Noldus system.

As stated above, MK-801 inhibits NMDA receptors and elicitshyperlocomotion. Therefore, where an antagonism of thehyperactivity-induced by the MK-801 is observed upon administration of acompound of the invention, it is an indication as to the positivemodulation of the NMDA activity. In other words, such a compound may beconsidered as useful in the treatment of schizophrenia.

Catalepsy

Catalepsy in mice and rats serves as a behavioral model for themanifestation of the extrapyramidal adverse effects of neuroleptics(Worm, et al., Dopamine-like activities of an aminopyridazinederivative, CM 30366. A behavioral study, Naunyn-Schnzeideberg's ArchPharmacol, 334, 246-52, 1986).

Catalepsy in mice is evaluated using a bar test. The mice were placed inthe middle of a steel rod situated between two platforms with theirfront paws resting on the bar. Animals were injected i.p. with a vehicleor Compound 1. Animals were scored 90 minutes later for the time it tookeach animal to reach the platform.

The Elevated Plus Maze

The elevated plus maze is a widely used method to test anxiety inrodents (Fellow, et al., Pharmacol. Biochem. Behav., 24, 525-529(1986)). The apparatus was made of wood and painted black, with twoopposing open arms and two opposite enclosed arms of the same size. Thearms were attached to a central square shaped in a plus sign. The wholeapparatus was placed 50 cm above the floor. Anxious animals refrain fromentering the open arm and prefer the closed arm. Benzodiazepines wereshown to increase the time spent in the open arms and the frequency ofentries to the open arms (Fellow, et al., Validation of open:closed armentries in an elevated plus-maze as a measure of anxiety in the rat, J.Neurosci. Methods, 14, 149-167 (1985)).

In a typical experiment, Balb/c male mice were administered orally with10 and 20 mg/kg of Compound 1 or Compound 6 and with a vehicle or withdiazepam (1 mg/kg) administered per os (p.o.) 90 minutes before theywere placed in the center of the maze. The frequency to the open arms,the time spent in the different arms, the velocity and number ofrearings of the animals in each zone were recorded (Noldus). Micetreated with a compound of the invention and exhibit preference ofentering to the open arms, or the center, and demonstrate a decrease inthe time spent in the closed arms, are indicative of anxiolytic activityimposed by the compound.

The Morris Water Maze

The Morris Water Maze is a well known test aimed to assess spatialcognitive tasks (Anger, et al., Animal test systems to study behavioraldysfunctions of neurodegenerative disorders, Neurotoxicology, 12, 403-13(1991)). The maze consists of a circular pool measuring 1.80 m indiameter 60 cm in height. The pool was filled with water (21±1° C.) to adepth of 30 cm. A circular hidden escape platform (10 cm in diameter)was placed just below the water surface. The test room contained severalpeitnanent extra maze cues such as the rat housing rack, laboratorytable, posters on the walls, etc.

C. Results and Discussion

The effect of Compound 1 on the behavior of mice in the open field andthe dose-dependent effect following i.p administration were evaluated.Naïve male Mice (Balb/c, Harlan Israel) were used. The animals, divided3 to 5 animals per group, were administered with Compound 1 (12.5, 25,50, 75 and 100 mg/kg) 1 hr prior to placing each in an open field. Micewere monitored for 1 hr (every 5 minutes) using the Noldus system, andtheir distance moved, velocity, time of immobility and number ofrearings was recorded.

As FIGS. 1A-D indicate, Compound 1 induced a dose dependent decrease inthe horizontal and vertical motility of the mice. At 12.5 mg/kg the drugdid not modify significantly neither parameter and tended to increaserearing frequency. Moreover, doses up to 50 mg/kg induced a mildinhibition of the horizontal and the vertical motility. Higher dosesshowed a marked sedative effect and motility was reduced to minimum.

The effect of Compound 6 on the behavior of mice in the open field wasalso examined. Naïve male mice (Balb/c, Harlan Israel) were used.Animals (6 per group) were administered with Compound 6 (12.5, 25 and 50mg/kg, i.p.) 1 hr prior to placing each in the open field. Mice werefollowed for 1 hr (every 5 minutes) using the Noldus system, and theirdistance moved, velocity, time of immobility and number of rearings wasrecorded.

As FIGS. 2A-D show, Compound 6 induced a dose dependent decrease in themotility of the mice expressed by decreased distance moved and velocity.The effect was mild up to 50 mg/kg. Compound 6 induced inhibition of thehorizontal (distance) and the vertical motility (number of rearings).The drug increased the duration of “not moving” periods at all doses. Itcan thus be concluded that Compound 6 as is the case with Compound 1 hasa mild sedative effect, as reflected by decreasing horizontal andvertical activity, mainly due to increased periods of immobility.

The effect of Compound 6 on the distance moved in Balb/c mice exposed toMK-801 (15 mg/kg) i.p. and to Compound 6 (up to 12.5 mg/kg, i.p.)administered 1 hr prior to MK-801 was also tested. The results indicatethat Compound 6 did not cause a consistent dose dependent effect on themotility of the MK-801 treated mice. The data suggests that Compound 6is a mild agent for the treatment of glutamate NMDA hypoactivity.

The effect of oral administration of Compound 1 in the open field wastested by orally administering to male ICR mice varying amounts ofCompound 1 (5, 10 and 20 mg/kg) or vehicle. Animals were placed in theopen field 1 hr after drug administration and horizontal and verticalbehavior was recorded for 20 minutes.

As FIGS. 3A-C show, oral administration of Compound 1 at 10 and 20 mg/kgslightly decreased the horizontal behavior as evidenced by the decreasein velocity and distance moved, and increased the vertical behavior asevidenced by increase in the number of rearings. It seems that thedecreased velocity and distance moved are related to more time that theanimal spent in the vertical position. The data also suggests thatCompound 1 induces a slight increase in vigilance and/or stimulation;this effect may also be relevant to a stimulatory effect on cognitionand learning.

The dose-dependent effect of Compound 3 administered i.p. in the openfield was also tested. Naïve male mice (Balb/c, Harlan Israel) wereused. Animals were administered with Compound 3 (12.5, 25 and 50 mg/kg,i.p., 6 per group) 1 hr prior to placing each in the open field. Micewere followed for 1 hr (every 5 minutes) using the Noldus system, andtheir distance moved, velocity, time of immobility and number ofrearings was recorded.

FIGS. 4A-C show the behavioral parameters of Compound 3 in the openfield. Compound 3 induced a mild decrease in the motility of the miceexpressed by decreased distance moved and velocity up to 25 mg/kg and amarked decrease in the motility parameters at 50 mg/kg (FIG. 4A). Thedrug dose dependently increased the duration of “not moving” periods(FIG. 4B) and decreased the number of rearings vs. controls (FIG. 4C).

Comparison Between Compound 1, Olanzapine or D-Serine on AnxietyParameters in Mice in the Open Field Paradigm

The effect of Compound 1, olanzapine and D-Serine [20 mg/kg, orally),administered 1 hr before placing the male ICR mice in the open field onanxiety parameters was examined in an open filed of the constructionshown above. Animals were administered orally with Compound 1 (5, 10 and20 mg/kg) or with a vehicle. Animals were placed in the open field 1 hrafter drug administration and animal track was registered for 20minutes. The time spent in the center (zone 3) and the frequency to thecenter were indicators of anxiety. High frequency and duration in thecenter indicated anxiolytic activity.

The results shown in FIG. 5 suggest that Compound 1 possesses anxiolyticactivity which differs from olanzapine or D-serine.

Amphetamine-Induced Hyperactivity and Stereotypic Behavior in Mice

As stated above, the amphetamine-induced hyperactivity and motility isone of the most popular animal models for schizophrenia. The experimentwas conducted with ICR male mice placed in individual barrels. Compound1 (20 mg/kg) was administered p.o to the mice 1 hr prior to i.p.administration of amphetamine (2 mg/kg). The hyper-active behaviorexpressed by the number of climbings and rearings and the stereotypicbehavior expressed by the number of head movements was recorded every 15minutes over a period of 1 h.

As FIGS. 6A-B show, Compound 1 was effective in antagonizinghyperactivity-induced by amphetamine. On the other hand, Compound 1alone increased the rearing behavior as compared to control animals.Similar results were obtained also in the open field experiment.Moreover, Compound 1 did not attenuate the increase in headmovements-induced by amphetamine. Overall, the data suggests a partialantagonist effect of Compound 1 at low doses against amphetamine-inducedhyperactivity. High efficacy was found in antagonizing increasedmotility, without affecting the stereotypic behavior. These data pointfor an efficacy of Compound 1 against psychotic symptoms, withoutinducing extrapyramidal symptoms.

The effect of Compound 3 (12.5, 25 and 50 mg/kg, i.p.) onamphetamine-induced hyperactivity in Balb/c mice was also examined.Animals (6 per group) were injected with Compound 3 (12.5, 25 and 50mg/kg) and 30 minutes later received amphetamine (2 mg/kg ip). After 30minutes, animals were exposed to an open field test for a period of 20minutes. Results show that Compound 3 dose-dependently antagonized theeffect of amphetamine and decreased hypermotility. Compound 3 combinedwith amphetamine dose-dependently increased immobility and was effectivealready at 12.5 mg/kg, i.p. This indicates that Compound 3 is highlyeffective against hyper-domaminergic activity as manifested byamphetamine treatment.

FIG. 7A shows the effect of Compound 3 (12.5, 25 and 50 mg/kg. i.p at±60 minutes) on distance moved. Results demonstrate a dose-dependentdecrease in the distance moved with normalization at 12.5 mg/kg and amarked decrease at higher doses. FIG. 7B shows the immobility time ofamphetamine and amphetamine combined with Compound 3. The results showthat amphetamine was similar to controls yet Compound 3 already at 12.5mg/kg antagonized the hyperactivity ensued by the amphetamine.

Overall, the results demonstrate a marked antidopaminergic activity forCompound 3, which may be useful for schizophrenia and related disorderstherapy.

Effect of Compound 1 on MK801-Induced Hyperactivity in Mice.

As stated above, MK-801 (an NMDA receptor antagonist) is a widelyaccepted animal model for schizophrenia. The experiment was conductedwith Balb/c male mice (Harlan Il), 6 per group. Compound 1 (50 mg/kg)was administered i.p. (1 hr before experimentation), to mice alone orcombined with MK-801 (0.15 mg/kg, i.p.) administered 20 minutes prior toplacing the mice individually in an open field for 60 minutes. Thehyper-active behavior expressed by hyperlocomotion, velocity, strongmobility, number of rearing was evaluated using the Noldus system, eachpoint represents the mean+/−6 determinations.

As FIGS. 8A-E show, at 50 mg/kg Compound 1 induces a clear cut decreasein both basal and MK-801-induced stimulation of horizontal motility asexpressed by distance moved, velocity, strong mobility (which isexpressed by moving the center of gravidance by more than 30%). Compound1 also markedly suppressed vertical motility (number of rearings) aloneand increased the inhibition of rearings presented by MK-801 as comparedto controls. This suggests that Compound 1 has a significant positivemodulation effect on the NMDA glutamate receptor.

As FIGS. 9A-B show, Compound 1 at 30 and 50 mg/kg, i.p. inhibited in adose related manner the hyperlocomotion effect of MK-801. FIGS. 10C andD show that Compound 1 at these concentrations did not modify theincreased frequency to the center of animals treated with MK-801, andeven increased the time spent in the center (zone 3). It can thus beconcluded that Compound 1 at high doses antagonizes positive symptoms ofMK-801 treated mice, without decreasing the anxiolytic activity of thedrug.

FIGS. 10A-D shows the effect of Compound 3 (12.5, 25 and 50 mg/kg, i.p.)on hyperactivity-induced by MK-801 (0.15 mg/kg, i.p.) in Balb/c mice.The data shows that Compound 3 induced a dose dependent decrease inMK-801-induced hyperactivity, a slight effect was observed with 7.5mg/kg, normalization of activity was achieved with 25 mg/kg and sedationwith 50 mg/kg (FIG. 10A). Immobility was not found with the small andintermediate doses (25 mg/kg) and appeared with the high dose (FIG.10B). Compound 3 at the low dose tended to increase the frequency to thecenter of the field (FIG. 10C) and the time spent in the center (FIG.10D). These results suggest a potential anxiolytic activity for Compound3.

Effect of Compound 1 at 30-50 mg/kg on Catalepsy in Mice

As FIG. 11 shows, Compound 1 at doses of up to 50 mg/kg did not inducecatalepsy in mice. This suggests that at doses which are efficaciousagainst positive symptoms of schizophrenia there is no catalepsy orextrapyramidal symptoms induced by Compound 1.

Effect of Compound 1 on the Forced Swim Test (FST)

Males Balb/c mice (Harlan Israel) were used. Animals were administeredorally with Compound 1 (10 or 20 mg/kg) or with a vehicle. The resultsshown in FIGS. 12A-D and FIGS. 13A-C demonstrate that orallyadministered Compound 1 at 10 mg/kg significantly increased the distancemoved (swimming behavior) and strong mobility (climbing behavior) and at20 mg/kg decreased immobility as compared to vehicle treated animals.The results suggest a significant antidepressant activity for Compound 1as appears in the forced swim test.

Comparison Between the Activity of Compound 1 and Olanzapine

Males ICR mice were used. Animals were administered orally witholanzapine or Compound 1 (20 mg/kg) or with a vehicle. The results shownin FIGS. 14A-B indicate that orally administered olanzapine at 20 mg/kgsignificantly decreased distance moved, velocity and strong mobility(climbing) and increased immobility. Compound 1 at the same dose did notmodify velocity and distance moved, and significantly decreased strongmobility (climbing) and immobility compared to vehicle and to olanzapinein treated animals. These data suggest that Compound 1 is different fromolanzapine, and at 20 mg/kg it causes decrease in immobility, suggestinga potential antidepressant activity.

Effect of Compound 1 on Anxiety in the Elevated Plus Maze

Male Balb/c mice were used. Animals were transferred to the behaviorroom 24 hr before the experiment. Compound 1 or diazepam wereadministered orally 90 minutes before testing. Each treatment groupincluded 5 animals. FIG. 15A shows the effect of Compound 1 on frequencyof the mice to the different arms. The results show that mice treatedwith Compound 1 (20 mg/kg) frequented significantly more the open armand the center resembling the mice treated with diazepam at 1 mg/kg.FIG. 15B shows the effect of Compound 1 on duration of time spent in thedifferent zones of the elevated plus maze. At 20 mg/kg Compound 1increased the time spent in the center, but did not significantly affectthe time spent in the closed and the open arm at variance from diazepam.FIG. 15C shows the velocity of the mice which was higher in the micetreated with diazepam but not in the mice treated with Compound 1.

FIG. 15D shows the frequency of rearings of mice treated with Compound1, diazepam or controls. The results show increased number of rearingsin the open arm in the diazepam and the Compound 1 treated groups. Asmay be noted, diazepam significantly increased the number of rearingsalso in the center.

From the above it may be concluded that at 20 mg/kg Compound 1 showedanxiolytic activity which resembles diazepam, but differs from it in itsintensity and in the effects on velocity.

FIG. 15E shows the total time spent by the mice administered withCompound 6 (3, 9 and 27 mg/kg, orally) on the different arms of themaze. The results demonstrate that mice treated with Compound 6 spentmore time on the open arms and less on the center, suggesting apotential anxiolytic activity for the Compound.

Effect of Compound 1 on Spatial Cognition in the Morris Water Maze

The Morris Water Maze is a well known test aimed to assess spatialcognitive tasks. The maze consists of a circular pool measuring 1.80 min diameter 60 cm in height. The pool was filled with water (21±1° C.)to a depth of 30 cm. A circular hidden escape platform (10 cm indiameter) was placed just below the water surface. The test roomcontained several permanent extra maze cues such as the rat housingrack, laboratory table, posters on the walls, etc.

In the experiment rats were given Compound 1 orally on day 1 and thefirst test begun 90 minute later, six trials per day, for 3 consecutivedays, to find the hidden platform (acquisition phase). The escapelatency, i.e., the time required by the rat to find and climb onto theplatform, was recorded for up to 120 sec. Each rat was allowed to remainon the platform for 30 sec, after which it was removed to its home cage.If the rat did not find the platform within 120 sec, it was manuallyplaced on it and returned to its home cage after 30 sec.

A video camera was placed above the center of the pool for tracking therat, and a video tracking system (Noldus) with online digital outputdirectly fed data into a computer. Data were analyzed using EthoVisionautomated tracking system software (Noldus).

FIG. 16 shows the effect of 10 and 20 mg/kg of Compound 1, administeredpo, on latency to platform. Compound 1 at 20 mg/kg showed a fasterlearning on the first day of the spatial task and reached the platformearlier. Also on the second day, an improvement was noticed of taskexecution by mice treated with Compound 1 (20 mg/kg). On the third day,no difference was found between all groups, and all rats reached rapidlythe platform. These results clearly suggest that Compound 1 can enhancecognitive tasks in the rats.

FIG. 17 shows the effect of acute use of Compound 1 at 10 and 20 mg/kggiven p.o. on day 1 on the spatial cognitive tasks in the Morris WaterMaze in rats pretreated (−30 minutes) with MK-801 (on day 1) at 0.15mg/kg, i.p. MK-801 alone induced a significant impairment in the memorytasks of the rats as expressed by increased latency to the platform ascompared to the normal controls. Compound 1 at both doses decreased thelatency to the platform on day 3 of the experiment but not on days 1 and2. These results may thus provide an indication as to the ability ofCompound 1 to relief some of the cognitive impairments observed instates of schizophrenia due to decreased NMDA activity.

The relief of cognitive impairments was also shown in the use ofCompound 6. FIG. 18 shows the effect of acute use of Compound 6 at 10mg/kg given p.o. once on day 1 on the spatial cognitive tasks in theMorris Water Maze in rats pretreated (−30 minutes) with MK-801 (0.15mg/kg, i.p.). MK-801 alone induced a significant impairment in thememory tasks of the rats as expressed by increased latency to theplatform as compared to the normal controls. Compound 6 at 10 mg/kgdecreased the latency to the platform on days 2 and 3 of theexperimentation. In similarity with Compound 1, the described use ofCompound 6 suggests use in relieving some of the cognitive impairmentsseen in states of schizophrenia due to decreased NMDA activity.

Overall and without wishing to be bound by theory, the effects ofCompound 1 and 6 on spatial memory suggest a common mechanism for botheffective activity on NMDA blockage and dopamine overshooting, andparallel stimulation of negative symptoms associated with cognitiveimpairment.

Effect of Compound 6 on Anxiety in the EPM Apparatus

Male Balb/c mice were used. Animals were transferred to the behaviorroom 24 hr before the experiment. Compound 6 (3, 9, or 27 mg/kg) orvehicle were administered orally 90 minutes before testing. Eachtreatment group included 5 animals. The effect of Compound 6 on durationof time spent in the different zones of the elevated plus maze isevident from the dose-dependently increase in the time spent in the openarms and decrease in the time spent in the center. The drug did notsignificantly affect the time spent in the closed arm. This suggeststhat Compound 6 has an anxiolytic activity.

Subchronic Toxicity

Subchronic toxicity was tested in ICR male mice. Animals (5 per group)received Compound 1 daily (10 and 20 mg/kg p.o). Animals were followedup for 7 days; weight, food intake and water intake were registered. Asshown in FIG. 19, no difference compared to control animals in bodyweight, food intake and water intake was found.

The drug was well tolerated, and no apparent difference was found alsoin animal general behavior. Further results showed that Compound 1 didnot cause toxic effect up to 20 mg/kg given for 7 days.

In-Vitro Toxicity

Since glutamate is known to possess a neurotoxic effect, the in-vitroeffect of Compound 1 was evaluated as compared to olanzapine, paroxetineand sertraline on human glioma U83 cell viability. Cells were treatedwith Compound 1, olanzapine, paroxetine, or sertraline as compared tocontrols (saline treated cells), 24 hr after exposure to the drug.Determination of cell viability was performed in cells (10,000/well)using neutral red method. Absorption of neutral red by lysosomes causedcoloring of living cells. Quantitative analysis was performed bycolorimetric assay (ELISA reader at 550 nm).

The results shown in FIG. 20 demonstrate that Compound 1 is not toxic upto 100 μM to human glioma cells. Olanzapine, sertraline and paroxetineat concentrations higher than 30 μM induced a dose dependent decrease incell viability. Overall, the results suggest that Compound 1 as theother compounds of the invention are well tolerated and possess lowtoxicity as presented by the in vivo and the in vitro assays detailedherein.

1. A compound, or a salt or stereoisomer thereof, of the formula L-M-V,wherein: L is olanzapine; M is optionally present and is a linkerselected from the group consisting of —NH—, —O—, —S—, C₁-C₈-alkylene,C₃-C₈-cycloalkylene, —CH₂—O—CH₂—, —(CH₂)_(n)—O—(CH₂)_(n)—,—(CH₂—O)_(n)—, and —(CH₂CH₂—O)_(n)—, said alkylene and cycloalkylenebeing optionally substituted by one or more group selected from thegroup consisting of C₁-C₄ alkyl, C₂-C₄ alkenyl, and C₂-C₄ alkynyl; V isa modulator of a glutamate N-methyl-D-aspartate (NMDA) receptor selectedfrom the group consisting of an amino acid, an ester of the amino acid,an amide of the amino acid, and an alkylated amine of the amino acid;wherein said amino acid is selected from the group consisting ofglycinyl, sarcosinyl, serinyl, and cysteinyl; and n, independently ofeach other, is an integer from 0 to
 3. 2. The compound, or a salt orstereoisomer thereof, according to claim 1, wherein bonds between M andL and between M and V are non-hydrolizable.
 3. The compound, or a saltor stereoisomer thereof, according to claim 1, wherein the compound isrepresented by formula (I):


4. The compound, or a salt or stereoisomer thereof, according to claim3, wherein V is glycinyl, or an ester thereof, and the compound is ofthe formula (Ia):

wherein M is optionally present and is selected from the groupconsisting of —NH—, —O—, —S—, C₁-C₈-alkylene, C₃-C₈-cycloalkylene,—CH₂—O—CH₂—, —(CH₂—O)_(n)—, and —(CH₂CH₂—O)_(n)—, and n is an integerfrom 1 to 3, and R is selected from the group consisting of H and C₁-C₄alkyl.
 5. The compound, or a salt or stereoisomer thereof, according toclaim 3, wherein V is selected from the group consisting of sarcosinylor an ester thereof and serinyl or an ester thereof.
 6. The compound, ora salt or stereoisomer thereof, according to claim 5, wherein thecompound is represented by formula (Ib):

wherein M is optionally present and is selected from the groupconsisting of —NH—, —O—, —S—, C₁-C₈-alkylene, C₃-C₈-cycloalkylene,—CH₂—O—CH₂—, —(CH₂—O)_(n)—, and —(CH₂CH₂—O)_(n)—, n is an integer from 1to 3, R and R′, independently of each other, are selected from the groupconsisting of H and C₁-C₄-alkyl, and R″ is selected from the groupconsisting of H and —CH₂OH.
 7. The compound, or a salt or stereoisomerthereof, according to claim 6, wherein M is absent, and R″ is H.
 8. Thecompound, or a salt or stereoisomer thereof, according to claim 6,wherein M is selected from the group consisting of —CH₂—O—CH₂—,—(CH₂—O)_(n)—, and —(CH₂CH₂—O)_(n)—, wherein n is an integer from 1 to3.
 9. The compound, or a salt or stereoisomer thereof, according toclaim 1, wherein the compound is selected from the following group:Compound No. L M V 1 Ola absent —CH(NH₂)(COOCH₃) 2 Ola absent—CH(NH₂)(COOCH₂CH₃) 3 Ola —OCH₂— —CH(NH₂)(COOCH₃) 4 Ola —OCH₂——CH(NH₂)(COOCH₂CH₃) 5 Ola absent —CH(NHCH₃)(COOH) 6 Ola absent—CH(NHCH₃)(COOCH₃) 7 Ola absent —CH(NHCH₃)(COOCH₂CH₃) 8 Ola absent—C(NH₂)(CH₂OH)(COOH) 9 Ola absent —C(NH₂)(CH₂OH)(COOCH₃) 10 Ola absent—C(NH₂)(CH₂OH)(COOCH₂CH₃) 11 Ola —OCH₂— —CH(NHCH₃)(COOH) 12 Ola —OCH₂——CH(NHCH₃)(COOCH₃) 13 Ola —OCH₂— —CH(NHCH₃)(COOCH₂CH₃) 14 Ola—CH₂CH₂OCH₂— —CH(NH₂)(COOCH₃) 15 Ola —CH₂CH₂OCH₂— —CH(NH₂)(COOCH₂CH₃) 16Ola —CH₂CH₂OCH₂— —CH(NHCH₃)(COOCH₃) 17 Ola —CH₂CH₂OCH₂——CH(NHCH₃)(COOCH₂CH₃).


10. A pharmaceutical composition comprising at least one compound, or asalt or stereoisomer thereof, according to claim 1, and a carrier.
 11. Amethod for treating schizophrenia, anxiety, and depression comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition according to claim
 10. 12. A method fortreating schizophrenia, said method comprising administering to a personsuffering from schizophrenia or symptoms associated therewith aneffective amount of a compound, or a salt or stereoisomer thereof,according to claim 1.